Photoprotective compositions containing malassezia-derived compounds and/or chemical analogs thereof

ABSTRACT

The present invention relates to compounds, compositions, and methods for modulating skin pigmentation and treating or preventing UV-induced skin damage, erythema, aging of the skin, sunburn, and hyperpigmentation in a subject. The compounds, compositions, and methods of the present invention generally involve Malassezia-derived compounds, including malassezin and indirubin, and/or chemical analogs thereof. Other applications of the compounds and compositions disclosed herein include, but are not limited to, improving hyperpigmentation caused by a hyperpigmentation disorder, inducing melanocyte apoptosis, and modulating arylhydrocarbon receptor (AhR) activity, melanogenesis, melanin production, melanosome biogenesis, melanosome transfer, melanocyte activity, and melanin concentration.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims benefit to U.S. provisional application No.62/722,412, filed Aug. 24, 2018, and U.S. provisional application No.62/742,657, filed Oct. 8, 2018. The entire contents of theaforementioned applications are incorporated by reference. Additionally,the entire contents of U.S. provisional application No. 62/306,468,filed Mar. 10, 2016, U.S. provisional application No. 62/656,769, filedApr. 12, 2018, U.S. provisional application No. 62/668,007, filed May 7,2018, U.S. provisional application No. 62/685,800, filed Jun. 15, 2018,U.S. provisional application No. 62/686,912, filed Jun. 19, 2018, U.S.patent application Ser. No. 15/455,932, filed Mar. 10, 2017, now U.S.Pat. No. 10,131,631, U.S. patent application Ser. No. 16/382,891, filedApr. 12, 2019, U.S. patent application Ser. No. 16/405,127, filed May 7,2019, and U.S. patent application Ser. No. 16/441,522, filed Jun. 14,2019 are hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates to compounds produced by or derived from aMalassezia yeast, as well as chemical analogs thereof. Compounds of thepresent invention, and compositions containing said compounds, have,among other beneficial properties, photoprotective properties. Methodsof using the compounds and compositions of the present invention arealso contemplated.

BACKGROUND OF THE INVENTION

Individuals around the world use skin brightening agents to achieve anumber of cosmetic goals, including producing an anti-aging effect,correcting sun damage, and meeting certain cultural standards of beauty.Many commercially available skin brightening products, while effectiveto varying degrees, contain harmful ingredients, some of which have beenlinked to cancer. Thus, there exists a need for novel skin brighteningagents and formulations that exhibit higher levels of safety and/orefficacy than agents currently on the market.

Malassezia is a genus of lipophilic yeast commonly found in the normalflora of human skin. Malassezia is responsible for a number of skindiseases, including Tinea versicolor (pityriasis versicolor), seborrheicdermatitis, and atopic dermatitis.

The natural habitat for M. furfur is the upper epidermis. However,exposure to ultraviolet light destroys the organism in its naturalhabitat. Therefore, UV filtering agents may be necessary for thesurvival of the organism. Two such UV-filtering indoles produced by theorganism have been identified: pityriacitrin and pityrialactone.Pityriacitrin, first described in Mayser et al., 2002, is synthesized byM. furfur. It is a stable yellow lipophilic compound showing broadabsorption in the UVA, UVB, and UVC spectrum. A similar compound fromthe genus Paracoccus has been isolated and patented as a UV protectiveagent. (Zhang et al., 2018).

Gambichler et al., 2007 investigated the UV protective effect ofpityriacitrin in humans using in vitro and in vivo test methods.Spectrophotometry of pityriacitrin cream and vehicle was performed inthe 290-400 nm wavelength range. UV transmission and the sun protectionfactor (“SPF”) were assessed for different cream formulations. Usingcolorimetry, the authors evaluated erythema and pigmentation followingirradiation of cream-protected and non-protected skin of healthysubjects. UVB as well as UVA transmission decreased with increasingpityriacitrin concentrations. An increase of pityriacitrin concentrationof 1.25, 2.5, and 5% was associated with slightly increasing SPFs of1.4, 1.5, and 1.7, respectively. The in vivo tests confirmed thevalidity of the SPF of pityriacitrin 5% cream determined in vitro.Overall, the UV protective effect of pityriacitrin was very weak,suggesting that pityriacitrin likely is only an inferior cofactor in thedevelopment of hypopigmentation in pityriasis versicolor alba lesionsfollowing sun exposure.

Further studies of the UV filtering effects of pityriacitrin wereperformed on human skin microflora. (Machowinski et al., 2006). Theauthors determined pityriacitrin has a UV-protective effect on Candidaalbicans and staphylococci with no toxicity in the ranges tested. The UVprotective properties of pityrialactone have also been confirmed in ayeast model. (Mayser et al., 2003). Pityrialactone appears to beresponsible for the yellow fluorescence of Tinea Versicolor under Wood'sLight examination.

Tinea versicolor is a non-contagious skin disease caused by Malasseziaovergrowth that locally alters pigmentation levels. Malassezia yeastshave two metabolic pathways for synthesizing melanin andtryptophan-derived indole pigments. Malassezin and Indirubin aretryptophan metabolites of Malassezia that may contribute to thedepigmentation characteristic of Malassezia overgrowth.

The invention disclosed herein utilizes compounds produced by or derivedfrom Malassezia yeast, including Malassezin, Indirubin, and chemicalanalogs thereof, as the basis for safe and efficacious skin brighteningand skin darkening compositions. Photoprotective compositions comprisingMalassezin, Indirubin, and chemical analogs thereof are also disclosedherein.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a composition. Thecomposition comprises one or more of the compounds listed in Table 1 orFIG. 3, or a chemical analog, crystalline form, hydrate, orpharmaceutically or cosmetically acceptable salt thereof.

Another embodiment of the present invention is a method for brighteningskin in a subject. The method comprises contacting the subject with oneor more of the compounds listed in Table 1 or FIG. 3, or a chemicalanalog, crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

A further embodiment of the present invention is a method for inducingmelanocyte apoptosis in a subject. The method comprises contacting thesubject with one or more of the compounds listed in Table 1 or FIG. 3,or a chemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof.

An additional embodiment of the present invention is a method formodulating arylhydrocarbon receptor (AhR) activity in a subject. Themethod comprises contacting the subject with one or more of thecompounds listed in Table 1 or FIG. 3, or a chemical analog, crystallineform, hydrate, or pharmaceutically or cosmetically acceptable saltthereof.

Another embodiment of the present invention is a method for modulatingmelanogenesis in a subject. The method comprises contacting the subjectwith one or more of the compounds listed in Table 1 or FIG. 3, or achemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof.

Another embodiment of the present invention is a method for modulatingmelanin concentration in a subject. The method comprises contacting thesubject with one or more of the compounds listed in Table 1 or FIG. 3,or a chemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof.

An additional embodiment of the present invention is a composition. Thecomposition comprises one or more of the compounds listed in Table 1 orFIG. 3, or a chemical analog, crystalline form, hydrate, orpharmaceutically or cosmetically acceptable salt thereof.

A further embodiment of the present invention is a composition forbrightening skin. The composition comprises one or more of the compoundslisted in Table 1 or FIG. 3, or a chemical analog, crystalline form,hydrate, or pharmaceutically or cosmetically acceptable salt thereof.

Another embodiment of the present invention is a composition forinducing melanocyte apoptosis. The composition comprises one or more ofthe compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

An additional embodiment of the present invention is a composition formodulating arylhydrocarbon receptor (AhR) activity. The compositioncomprises one or more of the compounds listed in Table 1 or FIG. 3, or achemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof.

A further embodiment of the present invention is a composition formodulating melanogenesis. The composition comprises one or more of thecompounds listed in Table 1 or FIG. 3, or a chemical analog, crystallineform, hydrate, or pharmaceutically or cosmetically acceptable saltthereof.

Another embodiment of the present invention is a composition formodulating melanin concentration. The composition comprises one or moreof the compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

An additional embodiment of the present invention is a method forbrightening skin in a subject. The method comprises contacting thesubject with a composition, the composition comprising one or more ofthe compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

A further embodiment of the present invention is a method for inducingmelanocyte apoptosis in a subject. The method comprises contacting thesubject with a composition, the composition comprising one or more ofthe compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

Another embodiment of the present invention is a method for modulatingarylhydrocarbon receptor (AhR) activity in a subject. The methodcomprises contacting the subject with a composition, the compositioncomprising one or more of the compounds listed in Table 1 or FIG. 3, ora chemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof.

An additional embodiment of the present invention is a method formodulating melanogenesis in a subject. The method comprises contactingthe subject with a composition, the composition comprising one or moreof the compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

A further embodiment of the present invention is a method for modulatingmelanin concentration in a subject. The method comprises contacting thesubject with a composition, the composition comprising one or more ofthe compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

Another embodiment of the present invention is a composition. Thecomposition comprises a Malassezia yeast and a cosmetically orpharmaceutically acceptable vehicle, diluent, or carrier.

An additional embodiment of the present invention is a composition. Thecomposition comprises a compound having the structure of the followingformula:

wherein:X is selected from the group consisting of NR₁₄ and O; Y is a covalentbond, CR₅R₆, O, or NR₁₅; R₁, R₂, R₃, R₄, R₇, R₈, R₉, R₁₀, and R₁₁ areindependently selected from the group consisting of hydrogen, halogen,CN, hydroxyl, R₁₆, or OR₁₆; R₁₃, R₁₄, and R₁₅ are independently hydrogenor R₁₆; R₅ and R₆ are independently selected from the group consistingof hydrogen, hydroxyl, OR₁₆, R₁₆, and C₃₋₆ cycloalkyl, or R₅ and R₆combine to form an oxo (═O) group or a C₃₋₆ cycloalkyl; R₁₂ is selectedfrom the group consisting of hydrogen, —COR^(a), and R₁₆, each R₁₆ isindependently formyl, C₁₋₉ alkyl, C₂₋₉ alkenyl, or C₂₋₉ alkynyl; and,R^(a) is selected from the group consisting of hydrogen, hydroxyl, andOR₁₆; or a crystalline form, hydrate, or cosmetically orpharmaceutically acceptable salt thereof, and a cosmetically orpharmaceutically acceptable vehicle, diluent, or carrier.

A further embodiment of the present invention is a composition. Thecomposition comprises a compound having the structure of the followingformula:

wherein:R₁, R₄, R₅, R₆, R₉, and R₁₀ are independently selected from the groupconsisting of hydrogen, hydroxyl, halogen, CN, R₁₃, OR₁₃, OCOR₁₃ and—CHO; R₂ and R₃ are independently selected from the group consisting ofhydrogen, hydroxyl, halogen, CN, R₁₃, OR₁₃, OCOR_(D) and —CHO, or R₂ andR₃ combine to form a 5- or 6-membered heterocyclyl; R₇ and R₈ areindependently selected from the group consisting of hydrogen, hydroxyl,halogen, CN, R₁₃, OR₁₃, OCOR_(D) and —CHO, or R₇ and R₈ combine to forma 5- or 6-membered heterocyclyl; R₁₁ and R₁₂ are independently hydrogenor R₁₃; and, each R₁₃ is independently C₁₋₉ alkyl, C₂₋₉ alkenyl, or C₂₋₉alkynyl; or a crystalline form, hydrate, or cosmetically orpharmaceutically acceptable salt thereof, and a cosmetically orpharmaceutically acceptable vehicle, diluent, or carrier.

Another embodiment of the present invention is a composition. Thecomposition comprises a compound listed in Table 1 or FIG. 3, or achemical analog, crystalline form, hydrate, or cosmetically orpharmaceutically acceptable salt thereof, and a cosmetically orpharmaceutically acceptable vehicle, diluent, or carrier.

An additional embodiment of the present invention is a method oftreating or preventing UV-induced skin damage in a subject. The methodcomprises contacting the subject with any of the compositions disclosedherein.

A further embodiment of the present invention is a method of treating orpreventing UV-induced erythema in a subject. The method comprisescontacting the subject with any of the compositions disclosed herein.

Another embodiment of the present invention is a method of treating orpreventing UV-induced aging of the skin in a subject. The methodcomprises contacting the subject with any of the compositions disclosedherein.

An additional embodiment of the present invention is a method oftreating or preventing sunburn in a subject. The method comprisescontacting the subject with any of the compositions disclosed herein.

A further embodiment of the present invention is a method of treating orpreventing UV-induced hyperpigmentation in a subject. The methodcomprises contacting the subject with any of the compositions disclosedherein.

Another embodiment of the present invention is a method for brighteningskin in a subject. The method comprises contacting the subject with anyof the compositions disclosed herein.

An additional embodiment of the present invention is a method forinducing melanocyte apoptosis in a subject. The method comprisescontacting the subject with any of the compositions disclosed herein.

A further embodiment of the present invention is a method for modulatingarylhydrocarbon receptor (AhR) activity in a subject. The methodcomprises contacting the subject with any of the compositions disclosedherein.

Another embodiment of the present invention is a method for modulatingmelanogenesis in a subject. The method comprises contacting the subjectwith any of the compositions disclosed herein.

An additional embodiment of the present invention is a method formodulating melanin concentration in a subject. The method comprisescontacting the subject with any of the compositions disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1-2 are tables showing mean tissue viability and melaninconcentration data ascertained from separate experiments withMelanoDerm™ substrates treated with varying concentrations of the testarticles shown.

FIG. 3 shows compounds produced by Malassezia.

FIGS. 4-5 are tables showing mean tissue viability and melaninconcentration data ascertained from separate experiments withMelanoDerm™ substrates treated with varying concentrations of the testarticles/test compositions shown.

FIGS. 6A-6B show synthesis schemes for AB17590 (FIG. 6A) and AB17653,AB17654, AB17655, AB17656, AB17657, and AB17658 (FIG. 6B).

FIG. 7 is a schematic showing a skin treatment template for Skin Type IVpatients. Values indicate UV dose for a given area in mJ/cm².

FIG. 8 is a table showing a Dualight scale for Skin Types I-VI.

FIG. 9 is a table showing Mexameter MX 16 measurements of melanin anderythema at Day 8 after Day 7 irradiation.

FIG. 10 is a table showing Mexameter MX 16 measurements of melanin anderythema at Day 15 after Day 14 irradiation.

FIG. 11 is a table showing an erythema scale of numerical valuesassociated with various degrees of erythema.

FIG. 12 is a photograph showing a subject's skin 24 hours afterirradiation with various levels of UV according to the skin treatmenttemplate shown in FIG. 7. The minimal erythema dose (“MED”) was 120 mJUVB 24 hours after irradiation.

FIG. 13 is a photograph showing test sites on a subject's skin at Day 7.

FIG. 14 is a photograph showing test sites on a subject's skin at Day 8,24 hours post-irradiation with 120 mJ UVB.

FIG. 15 is a photograph showing test sites on a subject's skin at Day 14after an additional week of Malassezin therapy. Treatment areas weredosed with 120 mJ UVB.

FIG. 16 is a photograph showing test sites on a subject's skin at Day15, 24 hours post-irradiation with 120 mJ UVB. Note erythema at vehiclesite for Days 7 and 9. Also note minimal to mild erythema at Malassezin1%-treated sites for Day 14, 10, and 8, with trace erythema at Days 1and 3.

DETAILED DESCRIPTION OF THE INVENTION

An additional embodiment of the present invention is a composition. Thecomposition comprises one or more of the compounds listed in Table 1 orFIG. 3, or a chemical analog, crystalline form, hydrate, orpharmaceutically or cosmetically acceptable salt thereof.

Another embodiment of the present invention is a composition forbrightening skin. The composition comprises one or more of the compoundslisted in Table 1 or FIG. 3, or a chemical analog, crystalline form,hydrate, or pharmaceutically or cosmetically acceptable salt thereof.

A further embodiment of the present invention is a composition forinducing melanocyte apoptosis. The composition comprises one or more ofthe compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

An additional embodiment of the present invention is a composition formodulating arylhydrocarbon receptor (AhR) activity. The compositioncomprises one or more of the compounds listed in Table 1 or FIG. 3, or achemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof.

Another embodiment of the present invention is a composition formodulating melanogenesis. The composition comprises one or more of thecompounds listed in Table 1 or FIG. 3, or a chemical analog, crystallineform, hydrate, or pharmaceutically or cosmetically acceptable saltthereof.

A further embodiment of the present invention is a composition formodulating melanin concentration. The composition comprises one or moreof the compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

An additional embodiment of the present invention is a method forbrightening skin in a subject. The method comprises contacting thesubject with a composition, the composition comprising one or more ofthe compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

Another embodiment of the present invention is a method for inducingmelanocyte apoptosis in a subject. The method comprises contacting thesubject with a composition, the composition comprising one or more ofthe compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

A further embodiment of the present invention is a method for modulatingarylhydrocarbon receptor (AhR) activity in a subject. The methodcomprises contacting the subject with a composition, the compositioncomprising one or more of the compounds listed in Table 1 or FIG. 3, ora chemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof.

An additional embodiment of the present invention is a method formodulating melanogenesis in a subject. The method comprises contactingthe subject with a composition, the composition comprising one or moreof the compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

Another embodiment of the present invention is a method for modulatingmelanin concentration in a subject. The method comprises contacting thesubject with a composition, the composition comprising one or more ofthe compounds listed in Table 1 or FIG. 3, or a chemical analog,crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.

In preferred embodiments, the compositions of the present inventioncomprise the compounds listed in Table 5.

In other preferred embodiments, the compositions of the presentinvention comprise the compounds listed in Table 6.

In additional preferred embodiments, the compositions of the presentinvention comprise the compounds listed in Table 7.

In further preferred embodiments, the compositions of the presentinvention comprise the compounds listed in Table 8.

In other preferred embodiments, the compositions of the presentinvention comprise the compounds listed in Table 9.

In additional preferred embodiments, the methods of the presentinvention comprise contacting a subject with a composition comprisingthe compounds listed in Table 5.

In further preferred embodiments, the methods of the present inventioncomprise contacting a subject with a composition comprising thecompounds listed in Table 6.

In other preferred embodiments, the methods of the present inventioncomprise contacting a subject with a composition comprising thecompounds listed in Table 7.

In additional preferred embodiments, the methods of the presentinvention comprise contacting a subject with a composition comprisingthe compounds listed in Table 8.

In further preferred embodiments, the methods of the present inventioncomprise contacting a subject with a composition comprising thecompounds listed in Table 9.

A further embodiment of the present invention is a composition. Thecomposition comprises a Malassezia yeast, and a cosmetically orpharmaceutically acceptable vehicle, diluent, or carrier.

An additional embodiment of the present invention is a composition. Thecomposition comprises a compound having the structure of the followingformula:

wherein:X is selected from the group consisting of NR₁₄ and O; Y is a covalentbond, CR₅R₆, O, or NR₁₅; R₁, R₂, R₃, R₄, R₇, R₈, R₉, R₁₀, and R₁₁ areindependently selected from the group consisting of hydrogen, halogen,CN, hydroxyl, R₁₆, or OR₁₆; R₁₃, R₁₄, and R₁₅ are independently hydrogenor R₁₆, R₅ and R₆ are independently selected from the group consistingof hydrogen, hydroxyl, OR₁₆, R₁₆, and C₃₋₆ cycloalkyl, or R₅ and R₆combine to form an oxo (═O) group or a C₃₋₆ cycloalkyl; R₁₂ is selectedfrom the group consisting of hydrogen, —COR^(a), and R₁₆, each R₁₆ isindependently formyl, C₁₋₉ alkyl, C₂₋₉ alkenyl, or C₂₋₉ alkynyl; and,R^(a) is selected from the group consisting of hydrogen, hydroxyl, andOR₁₆;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof,and a cosmetically or pharmaceutically acceptable vehicle, diluent, orcarrier.

Another embodiment of the present invention is a composition. Thecomposition comprises a compound having the structure of the followingformula:

wherein:R₁, R₄, R₅, R₆, R₉, and R₁₀ are independently selected from the groupconsisting of hydrogen, hydroxyl, halogen, CN, R₁₃, OR₁₃, OCOR_(D) and—CHO; R₂ and R₃ are independently selected from the group consisting ofhydrogen, hydroxyl, halogen, CN, R₁₃, OR₁₃, OCOR_(D) and —CHO, or R₂ andR₃ combine to form a 5- or 6-membered heterocyclyl; R₇ and R₈ areindependently selected from the group consisting of hydrogen, hydroxyl,halogen, CN, R₁₃, OR₁₃, OCOR_(D) and —CHO, or R₇ and R₈ combine to forma 5- or 6-membered heterocyclyl; R₁₁ and R₁₂ are independently hydrogenor R₁₃; and, each R₁₃ is independently C₁₋₉ alkyl, C₂₋₉ alkenyl, or C₂₋₉alkynyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof,and a cosmetically or pharmaceutically acceptable vehicle, diluent, orcarrier.

A further embodiment of the present invention is a composition. Thecomposition comprises a compound listed in Table 1 or FIG. 3, or achemical analog, crystalline form, hydrate, or cosmetically orpharmaceutically acceptable salt thereof, and a cosmetically orpharmaceutically acceptable vehicle, diluent, or carrier.

In preferred embodiments, any of the compositions of the presentinvention prevent UV-induced erythema in a subject.

In preferred embodiments, any of the compositions of the presentinvention reduce epidermal melanin in a subject.

In preferred embodiments, any of the compositions of the presentinvention produce a photo-protective or UV-protective effect in asubject.

In preferred embodiments, any of the compositions of the presentinvention filter, absorb, or reflect UV.

In preferred embodiments, any of the compositions of the presentinvention prevent hyperpigmentation and/or promote hypopigmentation.

In preferred embodiments, any of the compositions of the presentinvention is a sunscreening agent, a photo-protective agent, and/or aUV-protective agent.

An additional embodiment of the present invention is a method oftreating or preventing UV-induced skin damage in a subject. The methodcomprises contacting the subject with any of the compositions disclosedherein.

Another embodiment of the present invention is a method of treating orpreventing UV-induced erythema in a subject. The method comprisescontacting the subject with any of the compositions disclosed herein.

A further embodiment of the present invention is a method of treating orpreventing UV-induced aging of the skin in a subject. The methodcomprises contacting the subject with any of the compositions disclosedherein.

An additional embodiment of the present invention is a method oftreating or preventing sunburn in a subject. The method comprisescontacting the subject with any of the compositions disclosed herein.

Another embodiment of the present invention is a method of treating orpreventing UV-induced hyperpigmentation in a subject. The methodcomprises contacting the subject with any of the compositions disclosedherein.

A further embodiment of the present invention is a method forbrightening skin in a subject. The method comprises contacting thesubject with any of the compositions disclosed herein.

An additional embodiment of the present invention is a method forinducing melanocyte apoptosis in a subject. The method comprisescontacting the subject with any of the compositions disclosed herein.

Another embodiment of the present invention is a method for modulatingarylhydrocarbon receptor (AhR) activity in a subject. The methodcomprises contacting the subject with any of the compositions disclosedherein.

A further embodiment of the present invention is a method for modulatingmelanogenesis in a subject. The method comprises contacting the subjectwith any of the compositions disclosed herein.

An additional embodiment of the present invention is a method formodulating melanin concentration in a subject. The method comprisescontacting the subject with any of the compositions disclosed herein.

Definitions

As used herein, the term “compound” refers to two or more atoms that areconnected by one or more chemical bonds. In the present invention,chemical bonds include, but are not limited to, covalent bonds, ionicbonds, hydrogen bonds, and van der Waals interactions. Covalent bonds ofthe present invention include single, double, and triple bonds.Compounds of the present invention include, but are not limited to,organic molecules.

Organic compounds/molecules of the present invention include linear,branched, and cyclic hydrocarbons with or without functional groups. Theterm “C_(x-y)” when used in conjunction with a chemical moiety, such as,alkyl, alkenyl, alkynyl or alkoxy is meant to include groups thatcontain from x to y carbons in the chain. For example, the term “C_(x-y)alkyl” means substituted or unsubstituted saturated hydrocarbon groups,including straight-chain alkyl and branched-chain alkyl groups thatcontain from x to y carbons in the chain, including haloalkyl groupssuch as trifluoromethyl and 2,2,2-trifluoroethyl, and the like. Theterms “C_(x-y) alkenyl” and “C_(x-y) alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but containing atleast one double or triple bond, respectively.

The term “aliphatic”, as used herein, means a group composed of carbonand hydrogen atoms that does not contain aromatic rings. Accordingly,aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclylgroups.

As used herein, the term “alkyl” means acyclic linear and branchedhydrocarbon groups, e.g. “C₁-C₂₀ alkyl” refers to alkyl groups having1-20 carbons. An alkyl group may be linear or branched. Examples ofalkyl groups include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyltert-pentylhexyl, Isohexyl, and the like. Other alkyl groups will bereadily apparent to those of skill in the art given the benefit of thepresent disclosure. An alkyl group may be unsubstituted or substitutedwith one or more substituent groups as described herein. For example, analkyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or6 independently selected substituents) of halogen, —CO₂R′, —COOH, —CN,—OH, —OR′, —NH₂, —NHR′, —N(R′)₂, —SR′ or —SO₂R′, wherein each instanceof R′ independently is C₁-C₃ alkyl. In embodiments, the alkyl isunsubstituted. In embodiments, the alkyl is substituted (e.g., with 1,2, 3, 4, 5, or 6 substituent groups as described herein). For example,the term “hydroxyalkyl” refers to an alkyl group as described hereincomprising a hydroxyl (—OH) substituent and includes groups such as—CH₂OH.

As used herein, “alkenyl” means any linear or branched hydrocarbonchains having one or more unsaturated carbon-carbon double bonds thatmay occur in any stable point along the chain, e g “C₂-C₂₀ alkenyl”refers to an alkenyl group having 2-20 carbons. For example, an alkenylgroup includes prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl,hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. Inembodiments, the alkenyl comprises 1, 2, or 3 carbon-carbon doublebonds. In embodiments, the alkenyl comprises a single carbon-carbondouble bond. In embodiments, multiple double bonds (e.g., 2 or 3) areconjugated. An alkenyl group may be unsubstituted or substituted withone or more substituent groups as described herein. For example, analkenyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5,or 6 independently selected substituents) of halogen, —CO₂R′, —CN, —OH,—OR′, —NH₂, —NHR′, —N(R′)₂, —SR′ or —SO₂R′, wherein each instance of R′independently is C₁-C₃ alkyl. In embodiments, the alkenyl isunsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1,2, 3, 4, 5, or 6 substituent groups as described herein).

As used herein, “alkynyl” means any hydrocarbon chain of either linearor branched configuration, having one or more carbon-carbon triple bondsoccurring in any stable point along the chain, e.g. “C₂-C₂₀ alkynyl”refers to an alkynyl group having 2-20 carbons. Examples of an alkynylgroup include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl,3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like. Inembodiments, an alkynyl comprises one carbon-carbon triple bond. Analkynyl group may be unsubstituted or substituted with one or moresubstituent groups as described herein. For example, an alkynyl groupmay be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6independently selected substituents) of halogen, —CO₂R′, —CN, —OH, —OR′,—NH₂, —NHR′, —N(R′)₂, —SR′ or —SO₂R′, wherein each instance of R′independently is C₁-C₃ alkyl. In embodiments, the alkynyl isunsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1,2, 3, 4, 5, or 6 substituent groups as described herein).

As used herein, the term “cycloalkyl” means a nonaromatic, saturated,cyclic group, e.g. “C₃-C₁₀ cycloalkyl.” In embodiments, a cycloalkyl ismonocyclic. In embodiments, a cycloalkyl is polycyclic (e.g., bicyclicor tricyclic). In polycyclic cycloalkyl groups, individual rings can befused, bridged, or spirocyclic. Examples of a cycloalkyl group includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornanyl,bicyclo[3.2.1]octanyl, octahydro-pentalenyl, and spiro[4.5]decanyl, andthe like. The term “cycloalkyl” may be used interchangeably with theterm “carbocycle”. A cycloalkyl group may be unsubstituted orsubstituted with one or more substituent groups as described herein. Forexample, a cycloalkyl group may be substituted with one or more (e.g.,1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen,—CO₂R′, —CN, —OH, —OR′, —NH₂, —NHR′, —N(R′)₂, —SR′ or —SO₂R′, whereineach instance of R′ independently is C₁-C₃ alkyl. In embodiments, thecycloalkyl is unsubstituted. In embodiments, the cycloalkyl issubstituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups asdescribed herein).

As used herein, the term “halogen” means fluorine, chlorine, bromine, oriodine.

As used herein, an “aromatic compound”, “aromatic”, or compoundcontaining an “aromatic ring” is an aryl or a heteroaryl compound. Theterm “aryl” as used herein includes substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 3- to 8-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike. The term “heteroaryl” includes substituted or unsubstitutedaromatic single ring structures, preferably 3- to 8-membered rings, morepreferably 5- to 7-membered rings, even more preferably 5- to 6-memberedrings, whose ring structures include at least one heteroatom, preferablyone to four heteroatoms, more preferably one or two heteroatoms. Theterm “heteroaryl” also includes polycyclic ring systems having two ormore cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, indole,imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine,and pyrimidine, and the like. Preferably, certain compounds of thepresent invention include at least one, preferably two, indole groups aswell as at least one aldehyde group.

The term “substituted” means moieties having at least one substituentthat replaces a hydrogen atom on one or more carbons of the backbone. Itwill be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance with thepermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, and the like. The permissible substituents canbe one or more and the same or different for appropriate organiccompounds.

As used herein, the term “heterocycle” or “heterocyclic” means amonocyclic, bicyclic, or tricyclic ring system containing at least oneheteroatom. Heteroatoms include, but are not limited to, oxygen,nitrogen, and sulfur.

A monocyclic heterocyclic ring consists of, for example, a 3, 4, 5, 6,7, 8, 9, or 10-membered ring containing at least one heteroatom.Representative examples of monocyclic heterocyclic rings include, butare not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl,1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl,imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl,isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl,oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl,pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl,tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl,thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl(thiomorpholine sulfone), thiopyranyl, and trithianyl.

A bicyclic heterocyclic ring is, by non-limiting example, a monocyclicheterocyclic ring fused to a distal aryl ring or the monocyclicheterocyclic ring fused to a distal cycloalkyl ring or the monocyclicheterocyclic ring fused to a distal cycloalkenyl ring or the monocyclicheterocyclic ring fused to a distal monocyclic heterocyclic ring, or themonocyclic heterocyclic ring fused to a distal monocyclic heteroarylring. Representative examples of bicyclic heterocyclic rings include,but are not limited to, 1,3-benzodioxolyl, 1,3-benzodithiolyl,2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl,2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl, and1,2,3,4-tetrahydroquinolinyl.

A tricyclic heterocyclic ring is, by non-limiting example, a bicyclicheterocyclic ring fused to a phenyl group or the bicyclic heterocyclicring fused to a cycloalkyl group or the bicyclic heterocyclic ring fusedto a cycloalkenyl group or the bicyclic heterocyclic ring fused toanother monocyclic heterocyclic ring. Representative examples oftricyclic heterocyclic rings include, but are not limited to,2,3,4,4a,9,9a-hexahydro-1H-carbazolyl,5a,6,7,8,9,9a-hexahydrodibenzo[b,d]furanyl, and5a,6,7,8,9,9a-hexahydrodibenzo[b,d]thienyl.

Heterocycles of the present invention can be substituted withsubstituents independently selected from, by non-limiting example,alkenyl, alkoxy, alkoxyalkyl, alkoxyalkynyl, alkoxycarbonyl,alkoxycarbonylalkyl, alkoxy-NH═C(alkyl)-, alkyl, alkylcarbonyl,alkylcarbonylalkyl, alkylcarbonyloxy, alkylsulfonyl, alkylthio, alkynyl,aryl, arylalkoxy, arylalkyl, arylcarbonyl, aryloxy, carboxy,carboxyalkyl, cyano, cyanoalkyl, cycloalkyl, carbonyl, cycloalkylalkyl,formyl, halogen, haloalkyl, hydroxy, hydroxyalkyl, hydroxycycloalkyl,mercapto, nitro, oxo, and phenyl.

As used herein, “skin pigmentation modulating” and grammaticalvariations thereof refer generally to skin brightening as well as skindarkening effects of the compounds and compositions of the presentinvention.

As used herein, “skin brightening” and grammatical variations thereofrefer generally to any actual or perceived reduction in skinpigmentation. Skin brightening methods have been used to reducepigmentation of hyperpigmented areas of skin resulting from age, sunexposure, or a hyperpigmentation disorder. Application of the compoundsand compositions of the present invention to, for example, a subject'sskin, can reduce pigmentation so that the skin appears lighter or whiterthan before said application. Skin pigmentation can be assessed in anumber of ways, including, but not limited to, visual assessments using,for example, the von Luschan chromatic scale, the Fitzpatrick skintyping test (Fitzpatrick et al., 1988) and the Taylor HyperpigmentationScale (Taylor et al., 2005) and reflectance spectrophotometry methods(Zonios, et al., 2001). For example, the Fitzpatrick skin typing testincludes six types of skin (I-VI), and Type VI skin that becomes Type Vor less has been “brightened” as the term is used herein. As discussedfurther below, skin brightening can result due to a number of phenomena,including, but not limited to, modulation of melanocyte activity,induction of melanocyte apoptosis, or modulation of arylhydrocarbonreceptor (AhR) activity, melanogenesis, melanosome biogenesis,melanosome transfer, or melanin concentration.

Likewise, as used herein, “skin darkening” and grammatical variationsthereof refer generally to any actual or perceived increase in skinpigmentation. Skin darkening methods have been used to increasepigmentation of hypopigmented areas of skin resulting from, for example,a hypopigmentation disorder. Application of the compounds andcompositions of the present invention to, for example, a subject's skin,can increase pigmentation so that the skin appears darker than beforesaid application.

Certain compounds of the present invention are produced by, derivedfrom, isolated from, or isolatable from a Malassezia yeast. Malasseziayeasts are yeasts of the genus Malassezia and include, but are notlimited to, Malassezia globosa, Malassezia restricta, Malassezia furfur,Malassezia sympodialis, Malassezia slooffiae, Malassezia obtusa,Malassezia pachydermatis, Malassezia dermatis, Malassezia japonica,Malassezia nana, Malassezia yamatoensis, Malassezia equine, Malasseziacaprae, and Malassezia cuniculi. (Guého, et al., 1996; Gaitanis, et al.,2013). Malassezia yeast are part of the normal human cutaneous flora andtypically produce no pathogenic effects. However, Malassezia yeast cancause a number of diseases, including, but not limited to pityriasisversicolor (both the hyperpigmented and hypopigmented varieties),seborrheic dermatitis, dandruff, atopic dermatitis, Malasseziafolliculitis, psoriasis, and confluent and reticulated papillomatosis.(Gaitanis, et al., 2013).

As used herein, the term “chemical analog” refers to a compound that isstructurally related to a parent compound and contains differentfunctional groups or substituents. For example, parent compounds of thepresent invention include malassezin and indirubin, and chemical analogsof malassezin and indirubin contain certain functional groups andsubstituents that are distinct from malassezin and indirubin,respectively. Chemical analogs of the present invention may havesignificant advantages over a given parent compound, including apharmacokinetic profile suitable for cosmetic or pharmaceutical use. Insome embodiments, a chemical analog is generated from a parent moleculeby one or more chemical reactions. In other embodiments, alternativesynthesis schemes that do not originate with a parent compound can beused to generate chemical analogs of the present invention.

A compound of the present invention is produced by a Malassezia yeastif, over the course of its lifecycle, a Malassezia yeast wouldsynthesize, secrete, accumulate, or otherwise generate the compoundunder appropriate growth conditions. Malassezia yeast secrete differentcompounds depending on what their growth media is supplemented with.(Nazzaro-Porro, et al., 1978). The present invention includes anycompound produced by a Malassezia yeast under any growth condition, butpreferred compounds include, for example, malassezin, indirubin, andchemical analogs thereof.

A compound of the present invention is derived from a Malassezia yeastif, at any time over the course of the yeast's lifecycle, the compoundexisted on or in the yeast.

Malassezin is one example of a compound produced by a Malassezia yeastof the present invention. Malassezin, also known as2-(1H-indol-3-ylmethyl)-1H-indole-3-carbaldehyde, is a tryptophanmetabolite originally isolated from Malassezia furfur. Malassezin is aknown agonist of the arylhydrocarbon receptor (AhR), a receptorimplicated in cell growth, differentiation, and gene expression. (Willeet al., 2001). Malassezin also induces apoptosis in primary humanmelanocytes. (Kramer, et al., 2005). Recently, certain chemical analogsof malassezin were synthesized by Winston-McPherson and colleagues, whoexamined the analogs' AhR agonist activity. (Winston-McPherson, et al.,2014).

Indirubin is another example of a compound produced by a Malasseziayeast of the present invention. Indirubin is a metabolite isolated fromMalassezia furfur Indirubin is a known agonist of the arylhydrocarbonreceptor (AhR), a receptor implicated in cell growth, differentiation,and gene expression.

As used herein, the term “melanocyte” refers to a dendritic cell of theepidermis that normally synthesizes tyrosinase and, within melanosomes,the pigment melanin Melanocytes of the present invention exhibitupregulation of certain genes, including, but not limited to, one ormore of the following: tyrosinase (oculocutaneous albinism IA),microphthalmia-associated transcription factor, alpha-2-macroglobulin,tyrosinase-related protein 1, solute carrier family 16, GS3955 protein,v-kit Hardy-Zuckerman 4 feline sarcoma, ocular albinism 1, Rag Dprotein, glycogenin 2, G-protein-coupled receptor, family C,oculocutaneous albinism II, deleted in esophageal cancer 1, melan-A,SRY-box 10, ATPase, Class V, type 10C, matrix metalloproteinase 1,latent transforming growth factor beta b, ATP-binding cassette,sub-family C, hydroxyprostaglandin dehydrogenase 15, transmembrane 7superfamily member 1, glutaminyl-peptide cyclotransferase, and othergenes identified by Lee and colleagues. (Lee, et al., 2013).

Melanocytes, like many other cell types, undergo programmed cell deathor, apoptosis. Melanocyte apoptosis pathways are known to those of skillin the art (Wang, et al., 2014), and apoptosis pathways generally havebeen reviewed by Elmore (Elmore, 2007). A compound or composition of thepresent invention “induces” melanocyte apoptosis by, for example,causing the activation of certain pro-apoptotic signal transductionpathways or causing the repression of certain anti-apoptotic pathways ina melanocyte. It is envisioned that the compounds or compositions of thepresent invention can directly activate/repress an apoptosis-relatedpathway by directly interacting with a signaling molecule of the pathwayor by indirectly interacting with a molecule of the pathway via directinteraction with one or more intermediary molecules that do nottypically function within the pathway.

Melanocyte activity can be modulated in a number of ways contemplated inthe present invention, including, but not limited to, inducingmelanocyte apoptosis or altering melanocyte gene expression, cellmotility, cell growth, melanin production, melanosome biogenesis, ormelanosome transfer.

As used herein, the terms “modulate”, “modulating”, and grammaticalvariations thereof refer to an adjustment of a biological activity orphenomenon to a desired level. It is envisioned that “modulation” of thepresent invention includes adjustments that increase or decrease thelevels of the biological activity or phenomenon.

As used herein, the terms “agonist”, “agonizing”, and grammaticalvariations thereof refer to a molecule that triggers (e.g., initiates orpromotes), partially or fully enhances, stimulates or activates one ormore biological activities. Agonists of the present invention mayinteract with and activate a receptor, thereby inititating aphysiological or pharmacological response characteristic of thatreceptor. Agonists of the present invention include naturally occurringsubstances as well as synthetic substances.

As used herein, the terms “antagonist”, “antagonizing”, and grammaticalvariations thereof refer to a molecule that partially or fullysuppresses, inhibits, or deactivates one or more biological activities.Antagonists of the present invention may competitively bind to areceptor at the same site as an agonist, but does not activate theintracellular response initiated by the active form of the receptor.Antagonists of the present invention may inhibit intracellular responsesof an agonist or partial agonist.

An arylhydrocarbon receptor (AhR) of the present invention is anyarylhydrocarbon receptor that naturally exists in a subject as describedherein. Arylhydrocarbon receptors are known to those of skill in theart. (Noakes, 2015). Agonists of arylhydrocarbon receptors include, butare not limited to, tryptophan-related compounds such as kynurenine,kynurenic acid, cinnabarinic acid, and 6-formylindolo [3,2-1)] carbazole(FICZ). Malassezin is also known as an aryl hydrocarbon receptoragonist. (Wille, et al., 2001).

As used herein, the compounds, compositions, and methods of the presentinvention can be used to improve hyperpigmentation caused by ahyperpigmentation disorder by, for example, reducing the level ofhyperpigmentation in areas affected by a hyperpigmentation disorder,slowing further hyperpigmentation, or preventing furtherhyperpigmentation from occurring. However, because every subject may notrespond to a particular dosing protocol, regimen, or process, improvinghyperpigmentation caused by a hyperpigmentation disorder does notrequire that the desired physiologic response or outcome be achieved ineach and every subject or subject population. Accordingly, a givensubject or subject population may fail to respond or respondinadequately to dosing, but other subjects or subject populations mayrespond and, therefore, experience improvement in theirhyperpigmentation disorder.

As used herein, the term “hyperpigmentation” is an actual or a perceivedskin disorder of excessive dark color. The skin impairment can beactual, for example, attributed to age, excessive sun exposure, or adisease or condition leading to dark skin areas. The dark skin areas canbe in the form of spots, blotches, or relatively large areas of darkcolor. The skin impairment also can be perceived, for example, aperception by an individual that his/her skin shade is too dark. Theindividual may have a cosmetic desire to lighten the skin shade.

Hyperpigmentation disorders are disorders in which hyperpigmentation isthe primary symptom as well as disorders in which hyperpigmentationoccurs as a secondary symptom. Hyperpigmentation disorders of thepresent invention include, but are not limited to, congenitalhyperpigmentation disorders and acquired hyperpigmentation disorders.Congenital hyperpigmentation disorders of the present invention include,but are not limited to, those involving epidermal hyperpigmentation(nevus cell nevus, Spitz nevus, and nevus spilus), dermalhyperpigmentation (blue nevus, nevus Ohta, dermal melanosis, nevus Ito,and Mongolian spot), ephelides, acropigmentation reticularis,Spitzenpigment/acropigmentation, and lentiginosis (generalizedlentiginosis, LEOPARD syndrome, inherited patterned lentiginosis, Carneycomplex, Peutz-Jeghers syndrome, Laugier-Hunziker-Baran syndrome, andCronkhite-Canada syndrome). (Yamaguchi, et al., 2014). Acquiredhyperpigmentation disorders of the present invention include, but arenot limited to, senile lentigines/lentigo, melasma/chloasma, Riehl'smelanosis, labial melanotic macule, penile/vulvovaginal melanosis,erythromelanosis follicularis faciei Kitamura, UV-induced pigmentation(tanning and pigmentation petaloides actinica), postinflammatorypigmentation (friction melanosis and ashy dermatosis),chemical/drug-induced pigmentation (polychlorinated biphenyl, arsenic,5-FU, bleomycin, cyclophosphamide, methotrexate, chlorpromazine,phenytoin, tetracycline, and chloroquine), pigmentary demarcation lines,and foreign material deposition (such as carotene, silver, gold,mercury, bismuth, and tattoos). Hyperpigmentation related with systemicdisorders includes metabolism/enzyme disorders (hemochromatosis,Wilson's disease, Gaucher's disease, Niemann-Pick's disease,amyloidosis, ochronosis, acanthosis nigricans, and porphyria cutaneatarda), endocrine disorders (Addison's disease, Cushing syndrome, andhyperthyroidism), nutritional disorders (pellagra, vitamin B12deficiency, folic acid deficiency, vagabond's disease, and prurigopigmentosa), mastocytosis, collagen diseases, liver dysfunction, andkidney dysfunction. Hyperpigmentation can also be related withinfectious diseases (measles, syphilis, and Malassezia furfur) andsyndromes (von Recklinghausen's disease, Sotos syndrome, POEMS syndrome,Naegeli syndrome, Cantu syndrome, McCune-Albright syndrome, Watsonsyndrome, and Bloom syndrome). (Yamaguchi, et al., 2014).

Melanin is a naturally produced pigment that gives color to skin andhair. Melanin is produced by melanocytes in organelles known asmelanosomes by a process known as melanogenesis. A compound orcomposition of the present invention modulates melanin production (a/k/amelanogenesis) in a subject by, for example, modulating melanosomebiogenesis and directly or indirectly inhibiting melanin synthesis atthe enzymatic level.

Melanosome biogenesis occurs via four stages: Stage I is characterizedby pre-melanosomes, which are essentially non-pigmented vacuoles. Instage II, pre-melanosomes develop striations on which melanin isdeposited in stage III. Stage IV results in mature melanosomes that arerich in melanin content. Compounds and compositions of the presentinvention modulate melanosome biogenesis by inhibiting or attenuatingthe biological processes that normally promote any or all of thesestages. (Wasmeier, et al., 2008).

Melanin synthesis primarily involves three enzymes: tyrosinase,tyrosinase related protein-1, and dopachrome tautomerase. Additionalfactors that affect intracellular trafficking of these enzymes include,but are not limited to, BLOC-1, OA1, and SLC45A2. The compounds andcompositions of the present invention can modulate melanin productionby, for example, inhibiting or attenuating the activity of any of theseenzymes or factors. (Yamaguchi, et al., 2014).

Once melanosomes have formed and melanin has been synthesized,melanosomes need to be transferred from epidermal melanocytes to skinand hair keratinocytes. Melanosomes originate near the nucleus ofmelanocytes and are transported to the periphery of melanocytes alongmicrotubules and actin filaments. Compounds and compositions of thepresent invention modulate melanosome transfer by interfering with anyof the biological processes that result in the transport of melanosomesfrom the perinuclear region, to the melanocyte periphery, and intoadjacent keratinocytes.

Melanin concentration may be modulated by, for example, increasing ordecreasing melanogenesis or promoting melanin degradation in, orelimination from, a subject.

A compound isolated from a Malassezia yeast of the present inventionnecessarily exists, before isolation, in a Malassezia yeast or isproduced by a Malassezia yeast. Therefore, a compound isolated from aMalassezia yeast is derived from actual yeast cells. Standard protocolsfor extracting compounds from cellular material are known to those ofskill in the art.

A compound isolatable from a Malassezia yeast need not be derived fromactual yeast cells. Instead, synthetic reactions can be used to generatecompounds produced in yeast without the involvement of actual yeastcells. Organic synthesis reactions are well known to those of skill inthe art and can be used in this regard.

As used herein, the term “epidermal melanin” refers to melanin that isproduced in, transported to, or otherwise found in the epidermis.

As used herein, the term “reduce” and grammatical variations thereofmean to cause a decrease in the level of a given biological phenomenonor species. For example, compounds and compositions of the presentinvention reduce epidermal melanin in a subject, meaning that thecompounds and compositions of the present invention elicit a decrease inthe level of epidermal melanin in the subject. The term “reduce” andgrammatical variations thereof can mean, for example, decreasing thelevel of a given phenomenon or species by at least 5%, 10%, 25%, 50%,75%, or 100%.

As used herein, the term “contacting” and grammatical variations thereofrefer to bringing two or more materials into close enough proximity thatthey can interact. Thus, for illustrative purposes only, a compound ofthe present invention can contact a melanocyte by, for example,interacting with a receptor on the surface of the melanocyte. Similarly,a composition of the present invention can contact a human subject by,for example, being applied directly to the subject's skin.

As used herein, a “subject” means a mammalian cell, tissue, organism, orpopulations thereof. Subjects of the present invention are preferablyhuman, including human cells, tissues, and beings, but otherwiseinclude, primates, farm animals, domestic animals, laboratory animals,and the like. Some examples of agricultural animals include cows, pigs,horses, goats, and the like. Some examples of domestic animals includedogs, cats, and the like. Some examples of laboratory animals includeprimates, rats, mice, rabbits, guinea pigs, and the like.

As used herein, a subject “in need” of improvement in hyperpigmentationcaused by a hyperpigmentation disorder includes subjects with a real orperceived need of improvement.

As used herein, the terms “treat,” “treating,” “treatment” andgrammatical variations thereof mean subjecting an individual subject toa protocol, regimen, process or remedy, in which it is desired to obtaina physiologic response or outcome in that subject, e.g., a patient. Inparticular, the methods and compositions of the present invention may beused to slow the development of disease symptoms or delay the onset ofthe disease or condition, or halt the progression of diseasedevelopment. However, because every treated subject may not respond to aparticular treatment protocol, regimen, process or remedy, treating doesnot require that the desired physiologic response or outcome be achievedin each and every subject or subject population, e.g., patientpopulation. Accordingly, a given subject or subject population, e.g.,patient population may fail to respond or respond inadequately totreatment.

As used herein, the terms “prevent,” “preventing,” “prevention,” andgrammatical variations thereof mean that the compounds of the presentinvention are useful when administered to a patient who has not beendiagnosed as possibly having the disorder or disease at the time ofadministration, but who would normally be expected to develop thedisorder or disease or be at increased risk for the disorder or disease.The compounds and compositions of the invention, for example, slow thedevelopment of the disorder or disease symptoms, delay the onset of thedisorder or disease, or prevent the individual from developing thedisorder or disease at all. Preventing also includes administration ofthe compounds of the invention to those individuals thought to bepredisposed to the disorder or disease due to age, familial history,genetic or chromosomal abnormalities, and/or due to the presence of oneor more biological markers for the disorder or disease.

As used herein, the term “promote” and grammatical variations thereofmean to allow, enhance, permit, facilitate, foster, encourage, induce,or otherwise help to bring about.

As used herein, the term “produce” and grammatical variations thereofmean to cause a particular result to happen, occur, or come intoexistence. By non-limiting example, the compounds and compositions ofthe present invention produce a photoprotective or UV-protective effectin a subject.

As used herein, the term “erythema” refers to redness of the skin.Erythema may be caused by dilation and/or irritation of the superficialcapillaries. The term “UV-induced erythema” refers to skin redness thatdevelops as a result of UV exposure. As used herein, “sunburn” andgrammatical variations thereof refers to UV-induced erythema caused byexposure to sunlight or artificial UV sources (e.g. tanning beds).

As used herein, the term “hyperpigmentation” refers generally to an areaof skin wherein the pigmentation is greater than that of an adjacentarea of skin (e.g. a pigment spot, age spot, mole, and the like).Hyperpigmentation of the present invention includes, but is not limitedto, regional hyperpigmentation by melanocytic hyperactivity, otherlocalized hyperpigmentation by benign melanocytic hyperactivity andproliferation, disease-related hyperpigmentation, and accidentalhyperpigmentations such as those due to photosensitization, geneticmakeup, chemical ingestion, or other exposure (e.g. UV exposure), age,and post-lesional scarring. As used herein, “UV-inducedhyperpigmentation” refers to any hyperpigmentation caused by exposure tonatural or artificial UV.

As used herein, the term “hypopigmentation” refers generally to an areaof skin wherein the pigmentation is less than that of an adjacent areaof skin. Hypopigmentation of the present invention includes, but is notlimited to, vitiligo, depigmentation, Pityriasis alba, focalhypopigmentation, post-inflammatory hypopigmentation, piebaldism,albinism, Tinea versicolor, photosensitivity, leucism, hypomelanosis,atopic dermatitis, psoriasis, and the like.

As used herein, “UV-induced skin damage” means skin damage resultingfrom exposure to UV, including UVA, UVB, and UVC. UV-induced skin damageof the present invention includes, but is not limited to, wrinkles,hyperpigmentation, dysplasias, actinic keratosis, and skin cancers.

As used herein, “UV-induced aging of the skin” means skin agingresulting from exposure to UV, including UVA, UVB, and UVC. UV-inducedskin aging of the present invention manifests itself as, for example,wrinkles, fine lines, age spots, moles, dryness, thinness, or reducedelasticity of the skin, uneven skin tone, and other reductions in skinradiance, texture, resiliency, firmness, sagginess, and clarity caused,in whole or in part, by UV exposure.

As used herein, the term “photoprotective” and grammatical variationsthereof, when used to describe the effects of the compounds andcompositions of the present invention, mean that the compound andcompositions described herein prevent and/or mitigate damage caused bylight, particularly sunlight. Likewise, “photoprotective agents” of thepresent invention are those compounds and compositions described hereinthat prevent and/or mitigate damage caused by light, particularlysunlight.

As used herein, the term “UV-protective” and grammatical variationsthereof, when used to describe the effects of the compounds andcompositions of the present invention, mean that the compound andcompositions described herein prevent and/or mitigate damage caused byultraviolet (“UV”) light. Likewise, “UV-protective agents” of thepresent invention are those compounds and compositions described hereinthat prevent and/or mitigate damage caused by UV. Ultraviolet light ofthe present invention includes, for example, UVA (320-240 nm), UVB(290-320 nm), and UVC (200-290 nm).

As used herein, the term “filter” and grammatical variations thereofmean to block, reflect, absorb, or scatter UV. “Sunscreening agents” ofthe present invention include all compounds and compositions of thepresent invention that block, reflect, absorb, or scatter UV.

As used herein, the term “absorb” and grammatical variations thereofmean to take in UV or convert UV into heat energy. By non-limitingexample, compounds and compositions of the present invention can absorbUV and, as a result, radiate heat energy into their surroundings.

As used herein, the term “reflect” and grammatical variations thereof,when used in the context of UV, mean to throw or bounce UV back withoutabsorbing it.

As used herein, the term “composition” means an entity comprising one ormore compounds of the present invention, as well as any entity whichresults, directly or indirectly, from combinations of one or morecompounds of the present invention with other ingredients. Compositionsof the present invention can be used as, for example, in vifro or invivo research reagents. Compositions of the present invention can alsobe applied directly to the skin of a human or non-human subject for acosmetic or pharmaceutical effect. Additionally, compositions of thepresent invention comprise one or more of the compounds listed in Table1 or FIG. 3, or a chemical analog, crystalline form, hydrate, orpharmaceutically or cosmetically acceptable salt thereof.

A composition of the present invention may be administered in anydesired and effective manner for both in vifro and in vivo applications:for oral ingestion or for parenteral or other administration in anyappropriate manner such as intraperitoneal, subcutaneous, topical,intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual,intramuscular, intravenous, intraarterial, intrathecal, orintralymphatic. Further, a composition of the present invention may beadministered in conjunction with other compositions. A composition ofthe present invention may be encapsulated or otherwise protected againstgastric or other secretions, if desired.

The compositions of the invention comprise one or more activeingredients in admixture with one or more cosmetically orpharmaceutically acceptable carriers and, optionally, one or more othercompounds, ingredients and/or materials. Regardless of the route ofadministration selected, the compounds and compositions of the presentinvention are formulated into cosmetically or pharmaceuticallyacceptable dosage forms by conventional methods known to those of skillin the art.

Cosmetically or pharmaceutically acceptable vehicles, diluents andcarriers are well known in the art and include materials suitable forcontact with the tissues of humans and non-humans without unduetoxicity, incompatibility, instability, irritation, allergic responseand the like. Cosmetically or pharmaceutically acceptable vehicles,diluents and carriers include any substantially non-toxic substanceconventionally usable, for example, for topical, oral, peritoneal, orsubcutaneous administration of cosmetics or pharmaceuticals in which thecompounds and compositions of the present invention will remain stableand bioavailable when applied, ingested, injected, or otherwiseadministered to a human or non-human subject. Cosmetically orpharmaceutically acceptable carriers suitable for topical applicationare known to those of skill in the art and include cosmetically orpharmaceutically acceptable liquids, creams, oils, lotions, ointments,gels, or solids, such as conventional cosmetic night creams, foundationcreams, suntan lotions, sunscreens, hand lotions, make-up and make-upbases, masks and the like. Carriers suitable for a selected dosage formand intended route of administration are well known in the art, andacceptable carriers for a chosen dosage form and method ofadministration can be determined using ordinary skill in the art.

The compositions of the present invention can contain other ingredientsconventional in cosmetics including perfumes, estrogen, Vitamins A, Cand E, alpha-hydroxy or alpha-keto acids such as pyruvic, lactic orglycolic acids, lanolin, vaseline, aloe vera, methyl or propyl paraben,pigments and the like. Non-limiting cosmetically or pharmaceuticallyacceptable vehicles, diluents and carriers of the present inventioninclude sugars (e.g., lactose, sucrose, mannitol, and sorbitol),starches, cellulose preparations, calcium phosphates (e.g., dicalciumphosphate, tricalcium phosphate and calcium hydrogen phosphate), sodiumcitrate, water, aqueous solutions (e.g., saline, sodium chlorideinjection, Ringer's injection, dextrose injection, dextrose and sodiumchloride injection, lactated Ringer's injection), alcohols (e.g., ethylalcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol,propylene glycol, and polyethylene glycol), organic esters (e.g., ethyloleate and triglycerides), biodegradable polymers (e.g.,polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)),elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ,olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes(e.g., suppository waxes), paraffins, silicones, talc, silicylate, andthe like.

The compositions of the invention may, optionally, contain additionalingredients and/or materials commonly used in cosmetic compositions.These ingredients and materials are well known in the art and include,for example, (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, suchas glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose andsodium carbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,and sodium lauryl sulfate; (10) suspending agents, such as ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth; (11) buffering agents; (12) excipients, such as lactose,milk sugars, polyethylene glycols, animal and vegetable fats, oils,waxes, paraffins, cocoa butter, starches, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, andpolyamide powder; (13) inert diluents, such as water or other solvents;(14) preservatives; (15) surface-active agents; (16) dispersing agents;(17) control-release or absorption-delaying agents, such ashydroxypropylmethyl cellulose, other polymer matrices, biodegradablepolymers, liposomes, microspheres, aluminum monostearate, gelatin, andwaxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)emulsifying and suspending agents; (22), solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan; (23)propellants, such as chlorofluorohydrocathons and volatile unsubstitutedhydrocarbons, such as butane and propane; (24) antioxidants; (25) agentswhich render the formulation isotonic with the blood of the intendedrecipient, such as sugars and sodium chloride; (26) thickening agents;(27) coating materials, such as lecithin; and (28) sweetening,flavoring, coloring, perfuming and preservative agents. Each suchingredient or material must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Ingredients and materials suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable ingredients and materials for a chosen dosageform and method of administration may be determined using ordinary skillin the art.

Compositions of the present invention suitable for oral administrationmay be in the form of capsules, cachets, pills, tablets, powders,granules, a solution or a suspension in an aqueous or non-aqueousliquid, an oil-in-water or water-in-oil liquid emulsion, an elixir orsyrup, a pastille, a bolus, an electuary or a paste. These formulationsmay be prepared by methods known in the art, e.g., by means ofconventional pan-coating, mixing, granulation or lyophilizationprocesses.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared, e.g., bymixing the active ingredient(s) with one or more cosmetically orpharmaceutically acceptable carriers and, optionally, one or morefillers, extenders, binders, humectants, disintegrating agents, solutionretarding agents, absorption accelerators, wetting agents, absorbents,lubricants, and/or coloring agents. Solid compositions of a similar typemay be employed as fillers in soft and hard-filled gelatin capsulesusing a suitable excipient. A tablet may be made by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared using a suitable binder, lubricant, inertdiluent, preservative, disintegrant, surface-active or dispersing agent.Molded tablets may be made by molding in a suitable machine. Thetablets, and other solid dosage forms, such as capsules, pills andgranules, may optionally be scored or prepared with coatings and shells,such as enteric coatings and other coatings well known in the cosmeticformulating art. They may also be formulated so as to provide slow orcontrolled release of the active ingredient therein. They may besterilized by, for example, filtration through a bacteria-retainingfilter. These compositions may also optionally contain opacifying agentsand may be of a composition such that they release the active ingredientonly, or preferentially, in a certain portion of the gastrointestinaltract, optionally, in a delayed manner. The active ingredient can alsobe in microencapsulated form.

Liquid dosage forms for oral administration include cosmetically orpharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Compositions of the present invention for rectal or vaginaladministration may be presented as a suppository, which may be preparedby mixing one or more active ingredient(s) with one or more suitablenonirritating carriers which are solid at room temperature, but liquidat body temperature and, therefore, will melt in the rectum or vaginalcavity and release the active compound. Compositions of the presentinvention which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such cosmetically or pharmaceutically acceptable carriers asare known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops, emulsions, suspensions, aerosols, and inhalants. Anydesired conventional vehicles, assistants and optionally further activeingredients may be added to the formulation.

Preferred assistants originate from the group comprising preservatives,antioxidants, stabilisers, solubilisers, vitamins, colorants, odourimprovers, film formers, thickeners and humectants.

Solutions and emulsions can comprise the conventional vehicles, such assolvents, solubilisers and emulsifiers, for example water, ethanol,isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butyl glycol, oils, in particularcottonseed oil, groundnut oil, maize oil, olive oil, castor oil andsesame oil, glycerol fatty acid esters, polyethylene glycols and fattyacid esters of sorbitan, or mixtures of these substances.

The emulsions may exist in various forms. Thus, they can be, forexample, an emulsion or microemulsion of the water-in-oil (W/O) type orof the oil-in-water (O/W) type, or a multiple emulsion, for example ofthe water-in-oil-in-water (W/O/W) type.

The compositions according to the invention may also be in the form ofemulsifier-free, disperse preparations. They can be, for example,hydrodispersions or Pickering emulsions.

Suspensions may comprise conventional vehicles, such as liquid diluents,for example water, ethanol or propylene glycol, suspension media, forexample ethoxylated isostearyl alcohols, polyoxyethylene sorbitol estersand polyoxyethylene sorbitan esters, microcrystalline cellulose,aluminium metahydroxide, bentonite, agar-agar and tragacanth, ormixtures of these substances.

Pastes, ointments, gels and creams may comprise conventional vehicles,for example animal and vegetable fats, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures of thesesubstances.

Face and body oils may comprise the conventional vehicles, such assynthetic oils, such as fatty acid esters, fatty alcohols, siliconeoils, natural oils, such as vegetable oils and oily plant extracts,paraffin oils, lanolin oils, or mixtures of these substances.

Sprays may comprise the conventional propellants, for examplechlorofluorocarbons, propane/butane or dimethyl ether.

Compositions of the present invention suitable for parenteraladministrations comprise one or more compounds in combination with oneor more cosmetically or pharmaceutically acceptable sterile isotonicaqueous or non-aqueous solutions, dispersions, suspensions or emulsions,or sterile powders which may be reconstituted into sterile injectablesolutions or dispersions just prior to use, which may contain suitableantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient, or suspending or thickeningagents. Proper fluidity can be maintained, for example, by the use ofcoating materials, by the maintenance of the required particle size inthe case of dispersions, and by the use of surfactants. Thesecompositions may also contain suitable adjuvants, such as wettingagents, emulsifying agents and dispersing agents. It may also bedesirable to include isotonic agents. In addition, prolonged absorptionof the injectable cosmetic form may be brought about by the inclusion ofagents which delay absorption.

In some cases, in order to prolong the effect, it is desirable to slowits absorption from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline oramorphous material having poor water solubility.

The rate of absorption of the active agent/drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered composition may be accomplished by dissolvingor suspending the active composition in an oil vehicle. Injectable depotforms may be made by forming microencapsule matrices of the activeingredient in biodegradable polymers. Depending on the ratio of theactive ingredient to polymer, and the nature of the particular polymeremployed, the rate of active ingredient release can be controlled. Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue. Theinjectable materials can be sterilized for example, by filtrationthrough a bacterial-retaining filter.

The compositions of the present invention may be presented in unit-doseor multi-dose sealed containers, for example, ampules and vials, and maybe stored in a lyophilized condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the typedescribed above.

In the present invention, the term “crystalline form” means the crystalstructure of a compound. A compound may exist in one or more crystallineforms, which may have different structural, physical, pharmacological,or chemical characteristics. Different crystalline forms may be obtainedusing variations in nucleation, growth kinetics, agglomeration, andbreakage. Nucleation results when the phase-transition energy barrier isovercome, thereby allowing a particle to form from a supersaturatedsolution. Crystal growth is the enlargement of crystal particles causedby deposition of the chemical compound on an existing surface of thecrystal. The relative rate of nucleation and growth determine the sizedistribution of the crystals that are formed. The thermodynamic drivingforce for both nucleation and growth is supersaturation, which isdefined as the deviation from thermodynamic equilibrium. Agglomerationis the formation of larger particles through two or more particles(e.g., crystals) sticking together and forming a larger crystallinestructure.

The term “hydrate”, as used herein, means a solid or a semi-solid formof a chemical compound containing water in a molecular complex. Thewater is generally in a stoichiometric amount with respect to thechemical compound.

As used herein, “cosmetically or pharmaceutically acceptable salt”refers to a derivative of the compounds disclosed herein wherein thecompounds are modified by making acid or base salts thereof. Examples ofcosmetically or pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. For example, such salts include salts from ammonia,L-arginine, betaine, benethamine, benzathine, calcium hydroxide,choline, deanol, diethanolamine (2,2′-iminobis(ethanol)), diethylamine,2-(diethylamino)-ethanol, 2-aminoethanol, ethylenediamine,N-ethyl-glucamine, hydrabamine, 1H-imidazole, lysine, magnesiumhydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassiumhydroxide, 1-(2-hydroxy-ethyl)-pyrrolidine, sodium hydroxide,triethanolamine (2,2′,2″-nitrilotris(ethanol)), trometh-amine, zinchydroxide, acetic acid, 2.2-dichloro-acetic acid, adipic acid, alginicacid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoicacid, 2,5-dihydroxybenzoic acid, 4-acetamido-benzoic acid, (+)-camphoricacid, (+)-camphor-10-sulfonic acid, carbonic acid, cinnamic acid, citricacid, cyclamic acid, decanoic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid,2-hydroxy-ethanesulfonic acid, ethylenediamonotetraacetic acid, formicacid, fumaric acid, galacaric acid, gentisic acid, D-glucoheptonic acid,D-gluconic acid, D-glucuronic acid, glutamic acid, glutantic acid,glutaric acid, 2-oxo-glutaric acid, glycero-phosphoric acid, glycine,glycolic acid, hexanoic acid, hippuric acid, hydrobromic acid,hydrochloric acid isobutyric acid, DL-lactic acid, lactobionic acid,lauric acid, lysine, maleic acid, (−)-L-malic acid, malonic acid,DL-mandelic acid, methanesulfonic acid, galactaric acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, octanoic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid(embonic acid), phosphoric acid, propionic acid, (−)-L-pyroglutamicacid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearicacid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid,thiocyanic acid, p-toluenesulfonic acid and undecylenic acid. Furthercosmetically or pharmaceutically acceptable salts can be formed withcations from metals like aluminum, calcium, lithium, magnesium,potassium, sodium, zinc and the like.

The cosmetically or pharmaceutically acceptable salts of the presentinvention can be synthesized from a compound disclosed herein whichcontains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with a sufficient amount of the appropriatebase or acid in water or in an organic diluent like ether, ethylacetate, ethanol, isopropanol, or acetonitrile, or a mixture thereof.

It is envisioned that the compounds and compositions of the presentinvention may be included in cosmetic or pharmaceutical compositions forboth in vitro and in vivo applications.

It is envisioned that the compounds and compositions of the presentinvention, including one or more compounds listed in Table 1 or FIG. 3,or a chemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof, may be co-administered to asubject to effectuate the skin pigmentation-modulating purposes of thepresent invention.

It is also envisioned that the compositions of the present invention maycomprise one or more compounds listed in Table 1 or FIG. 3, or achemical analog, crystalline form, hydrate, or pharmaceutically orcosmetically acceptable salt thereof. For example, a composition of thepresent invention may comprise indirubin or chemical analogs thereof incombination with malassezin or chemical analogs thereof.

Additionally, it is envisioned that the compounds of the presentinvention include compounds produced by Malassezia, or a chemicalanalog, crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof. Further, it is envisioned that the compositionsand methods of the present invention may involve one or more compoundsproduced by Malassezia, or a chemical analog, crystalline form, hydrate,or pharmaceutically or cosmetically acceptable salt thereof. Forexample, compounds produced by, or derived from, Malassezia include, butare not limited to, the compounds shown in FIG. 3.

It is further envisioned that the methods of the present invention mayinvolve co-administering two or more compounds and/or compositions ofthe present invention to effectuate the skin pigmentation-modulatingpurposes described herein.

Co-administered compounds and compositions of the present invention may,for example, contact a subject at substantially the same time or oneafter another.

The compositions of the present invention containing one or moreMalassezia-derived compounds or chemical analogs thereof may demonstratesynergistic effects over component compounds alone on various efficacycriteria, including, but not limited to, mean tissue viability, melaninconcentration, skin brightening, skin darkening, induction of melanocyteapoptosis, and modulation of arylhydrocarbon (AhR) activity,melanogenesis, or melanin concentration.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitlycontemplated.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Compound Designations

Table 1 below shows structures and names for compounds of the instantinvention.

TABLE 1 Compound Code Compound Name Structure CV-8684 Malassezin

N/A Malassezin Precursor

CV-8685 Indolo[3,2-b] carbazole

CV-8686 Compound I

CV-8687 Compound IV

CV-8688 Compound II

CV-8802 Compound C

CV-8803 Compound K

CV-8804 Compound A

AB12508 Compound E

CV-8819 Compound A5

AB12509 Compound H

CV-8877 Compound B

N/A Compound B10

AB11644 N/A

AB12976 O52

AB17011 Malassezia Indole A

AB17014 Pityriacitrin

AB17151 N/A

AB17225 Compound IV

AB17227 Malassezialactic Acid

AB12507 N/A

AB17219 Compound V

N/A FICZ

AB17220 Compound VIII

AB17221 Compound VII

N/A Indirubin

AB17590 N/A

AB17653 N/A

AB17654 N/A

AB17655 N/A

AB17656 N/A

AB17657 N/A

AB17658 N/A

N/A Compound C1

N/A Compound C2

Example 2 Apoptosis-Inducing Activity of Indirubin and IndirubinDerivatives Reagents

Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit, Fetal Bovine Serum(FBS), 0.25% Trypsin-EDTA (lx), Caspase-Glo 3/7 Assay, RPMI 1640 Medium,Dulbecco's Modified Eagle Medium, and Antibiotic Antimycotic Solution(100×).

The cell lines MeWo (ATCC® HTB-65™), WM115 (ATCC® CRL-1675) and B16F1(ATCC® CRL-6323) are maintained in the following culture media: culturemedium for MeWo and B16F1: DMEM supplemented with 10% FBS; culturemedium for WM115: RPMI 1640 supplemented with 10% FBS.

Experimental Methods

Cells are harvested and the cell number determined using a Countess CellCounter. The cells are diluted with culture medium to the desireddensity. The final cell density may be, for example, 4,000 cells/wellfor 6 hr and 24 hr treatment, and 2,000 cells/well for 48 hr and 72 hrtreatment. For the Annexin V assay, 384-well clear-bottom plates(Corning 3712) are employed, whereas 384-well solid white-bottom plates(Corning 3570) are used for the Caspase-Glo assays. All plates arecovered with a lid and placed at 37° C. and 5% CO₂ overnight for cellattachment.

Test compounds are dissolved in DMSO to 30 mM stock. 10-fold dilutionsare performed to generate 3 mM and 0.3 mM concentrations. 0.9 mMStaurosporine is employed as positive control, and DMSO is employed asnegative control (NC). 132.5 nL of compounds is transferred fromcompound source plate to 384-well cell culture plate(s) using liquidhandler Echo550. After the indicated incubation time, the plates areremoved from the incubator for detection.

For the Annexin V assay, plates are removed from the incubator andculture media is removed. Cells are washed twice with 40 uL PBS and 15uL of pre-mixed Annexin V-FITC and Hoechst 33342 dye working solutionare added per well. Plates are incubated at room temperature for 20minutes, sealed, and centrifuged for 1 minute at 1,000 rpm to removebubbles. Plates are read using ImageXpress Nano.

For the Caspase-Glo assay, plates are removed from the incubator andequilibrated at room temperature for 15 minutes. Caspase-Glo 3/7reagents also are thawed and equilibrated to room temperature before theexperiment. Caspase-Glo reagent is added to the required wells at 1:1ratio to the culture medium. Plates are incubated at room temperaturefor 15 minutes and read using EnSpire™ plate reader. Fold induction iscalculated according to the following formula: Foldinduction=Lum_(Sample)/Lum_(NC).

Annexin V Assay and Caspase 3/7 Assay Results

It is expected that the compounds and compositions of the presentinvention, including indirubin and chemical analogs thereof, will inducecell death. Chemical analogs of indirubin are expected to exhibit, forexample, more potent apoptosis-inducing activity compared to indirubin.Likewise, certain chemical analogs of indirubin are expected todemonstrate, for example, less effective apoptosis-inducing activitycompared to indirubin. Such compounds may have more favorable toxicityprofiles compared to more potent compounds.

Example 3 Cell Viability After Exposure to Indirubin and IndirubinDerivatives Reagents

CellTiter-Glo® 2.0 assay.

Experimental Methods

For the CellTiter-Glo assay, test compounds are prepared in 10 mM DMSOsolution. Compounds are serially diluted into 12 concentrations. 40 uLof cells from a 100,000 cell/mL suspension are dispensed into each wellof a 384-well plate (Corning 3570). Plates are incubated overnight at37° C., 5% CO₂, and 95% humidity. Test compounds are added, with DMSO asvehicle control. Plates are incubated at 37° C., 5% CO₂, and 95%humidity for 6, 24, or 48 hours, and 40 uL of CellTiter-Glo reagent isadded to the wells to assess cell viability.

Results

It is expected that the compounds and compositions of the presentinvention, including indirubin and chemical analogs thereof, will inducecell death. Chemical analogs of indirubin are expected to exhibit, forexample, more potent apoptosis-inducing activity compared to indirubin.Likewise, certain chemical analogs of indirubin are expected todemonstrate, for example, less effective apoptosis-inducing activitycompared to indirubin. Such compounds may have more favorable toxicityprofiles compared to more potent compounds.

Example 4 Arylhydrocarbon Receptor Activation Potential of Indirubin andIndirubin Derivatives Assay Procedures

Culture media for stably transfected HepG2 cells is prepared bysupplementing DMEM with high glucose and L-glutamine, as well as 10%FBS.

HepG2-AhR-Luc cells are cultured in T-75 flasks at 37° C., 5% CO₂, and95% relative humidity. Cells are allowed to reach 80-90% confluencebefore detachment and splitting.

Cultivated cells are rinsed with 5 mL PBS. PBS is aspirated away, 1.5 mLtrypsin is added to the flask, and cells are incubated at 37° C. forapproximately 5 minutes or until the cells are detached and float.Trypsin is inactivated by adding excess serum-containing media.

The cell suspension is transferred to a conical tube and centrifuged at120 g for 10 minutes to pellet the cells. Cells are resuspended inseeding media at a proper density. 40 μL of cells are transferred to a384-well culture plate (5×10³ cells/well). Plates are placed in theincubator at 37° C. for 24 hours.

Afterward, stock solutions of test compounds and omeprazole positivecontrol are prepared. Compound solutions are transferred into the assayplate using Echo550. The plate is then placed back into the incubatorfor compound treatment.

Later, after 24 hours of treatment, the plate is removed from theincubator and allowed to cool at ambient temperature. 30 μL One-Gloreagent equal to that of the culture medium is added in each well. Cellsare allowed to lyse for at least 3 minutes, and then measured in aluminometer.

Dose responses are graphed using the non-linear regression analysis inXLfit, and EC₅₀ values are also calculated.

Results

It is expected that the compounds and compositions of the presentinvention, including indirubin and chemical analogs thereof, willmodulate AhR activity. Chemical analogs of indirubin are expected toexhibit, for example, more potent AhR agonist activity compared toindirubin. Likewise, certain chemical analogs of indirubin are expectedto demonstrate, for example, less effective AhR agonist activitycompared to indirubin.

Example 5 MelanoDerm™ Assays

The purpose of this study was to evaluate the potential action of thetest articles as a skin melanogenesis modulator in the MelanoDerm™ SkinModel after repeated test article exposures. Secondarily, the purpose ofthis study was to evaluate the potential dermal irritation of the testarticle to the MelanoDerm™ Skin Model after repeated exposures. Toxicitywas determined by measuring the relative conversion of MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in thetest article-treated tissues compared to the negative/solventcontrol-treated tissues. The potential impact on melanin production wasdetermined by measuring the concentration of melanin produced by thetest article-treated tissues compared to the negative/solventcontrol-treated tissues.

Identification of Test Substances and Assay Controls

TABLE 2 Test Articles Tested in Diluted Form Test Article Sponsor DosingDesignation Designation Concentration Preparation Instructions 17AA70DMSO (solvent 0.5% (v/v) The test article was diluted (v/v) withEPI-100- control) LLMM to a final concentration of 0.5%; the dilutedtest article was vortexed for at least 1 minute and dosed onto thetissues using a dosing volume of 25 μL. A total volume of ~0.5 mL wasprepared for each tissue treatment. 17AD45 Compound K 500 μM Startingfrom the stock concentration provided, (CV-8803) the test article wasdiluted (v/v) with EPI-100- 17AJ41 Malassezin 500 μM LLMM to the finalconcentration of 500 μM. The (CV-8684) test article dilution wasvortexed for at least 1 17AJ43 Compound B 500 μM minute, heated at 37° ±1° C. (in a water bath) for (CV-8877) 15 minutes, vortexed again for atleast 1 minute 17AJ44 Compound E 500 μM and dosed on the tissues using adosing volume (AB12508) of 25 μL. A total volume of ~0.5 mL was 18AA14AB17151 500 μM prepared for each tissue treatment. 18AD42 Indirubin 500μM Starting from the solid material provided, a stock solution of ~100mM was prepared in DMSO. The stock dilution was stored at −15° C. to−25° C. From the stock concentrations thus prepared, the test articlewas further diluted with EPI-100- LLMM to the final concentration of 500μM. The test article dilution was vortexed for at least 1 minute, heatedat 37° ± 1° C. (in a water bath) for 15 minutes, vortexed again for atleast 1 minute and dosed on the tissues using a dosing volume of 25 μL.A total volume of ~0.5 mL was prepared for each tissue treatment.

TABLE 3 Test Articles Tested As Combinations Test Article Sponsor DosingDesignation Designation Concentration Preparation Instructions 17AJ41Malassezin (CV- 250 μM A total volume of ~1.0 mL of the combined test8684) article was prepared for each tissue treatment as 18AD42 Indirubin250 μM follows: 2 μL of 17AJ41 (100 mM) 2 μL of 18AD42 (100 mM) 796 μLof EPI-100-LLMM The test article combination was vortexed for at least 1minute, heated at 37° ± 1° C. (in a water bath) for 15 minutes, vortexedagain for at least 1 minute and dosed on the tissues using a dosingvolume of 25 μL. 18AD42 Indirubin 250 μM A total volume of ~1.0 mL ofthe combined 18AA14 AB17151 250 μM test article was prepared for eachtissue treatment as follows: 2 μL of 18AD42 (100 mM) 2 μL of 18AA14 (100mM) 796 μL of EPI-100-LLMM The test article combination was vortexed forat least 1 minute, heated at 37° ± 1° C. (in a water bath) for 15minutes, vortexed again for at least 1 minute and dosed on the tissuesusing a dosing volume of 25 μL. 17AJ44 Compound E 100 μM A total volumeof ~1.0 mL of the combined (AB12508) test article was prepared for eachtissue 17AJ43 Compound B 100 μM treatment as follows: (CV-8877) 1 μL of17AJ44 (100 mM) 1 μL of 17AJ43 (100 mM) 998 μL of EPI-100-LLMM The testarticle combination was vortexed for at least 1 minute, heated at 37° ±1° C. (in a water bath) for 15 minutes, vortexed again for at least 1minute and dosed on the tissues using a dosing volume of 25 μL. 17AJ43Compound B 100 μM A total volume of ~1.0 mL of the combined (CV-8877)test article was prepared for each tissue 18AA14 AB17151 100 μMtreatment as follows: 1 μL of 17AJ43 (100 mM) 1 μL of 18AA14 (100 mM)998 μL of EPI-100-LLMM The test article combination was vortexed for atleast 1 minute, heated at 37° ± 1° C. (in a water bath) for 15 minutes,vortexed again for at least 1 minute and dosed on the tissues using adosing volume of 25 μL.

Assay controls include: positive control—1% Kojic Acid; negativecontrol—sterile, deionized water; and solvent control—DMSO (dimethylsulfoxide) prepared in EPI-100-LLMM.

For this study, a negative control was not used. Instead, the solventcontrol (17AA70) was used to correct the data pertaining to the positivecontrol- and test article-treated tissues, respectively.

Additionally, the test article and controls were applied to groups of 4tissues of which 2 were used for the Tissue Viability (MTT) endpoint and2 for the Melanin endpoint, respectively.

Test System

The MelanoDerm™ Skin Model provided by MatTek Corporation (Ashland,Mass.) was used in this study. The MelanoDerm™ tissue consists ofnormal, human-derived epidermal keratinocytes (NHEK) and melanocytes(NHM) which have been cultured to form a multilayered, highlydifferentiated model of the human epidermis. The NHMs within co-culturesundergo spontaneous melanogenesis leading to tissues of varying levelsof pigmentation. The cultures were grown on cell culture inserts at theair-liquid interface, allowing for topical application of skinmodulators. The MelanoDerm™ model exhibits in vivo-like morphologicaland ultrastructural characteristics. NHM localized in the basal celllayer of MelanoDerm™ tissue are dendritic and spontaneously producemelanin granules which progressively populate the layers of the tissue.Thus the test system is used to screen for materials which may inhibitor stimulate the production of melanin relative to the negativecontrols.

Experimental Design and Methodology

The experimental design of this study consisted of the determination ofthe pH of the neat test article if possible (and/or dosing solution asappropriate) and a definitive assay to determine the relative tissueviability and the potential action of the test article as a skinmelanogenesis modulator to MelanoDerm™ Skin Model after repeatedexposures. The test articles were exposed to the MelanoDerm™ Skin Modelfor a total of 7 days. The test articles were topically applied to theMelanoDerm™ Skin Model every 48 hours (within a timeframe of 48+2 hoursfrom previous treatment). The toxicity of the test articles weredetermined by the NAD(P)H-dependent microsomal enzyme reduction of MTT(and, to a lesser extent, by the succinate dehydrogenase reduction ofMTT) in control and test article-treated tissues. Data was presented inthe form of relative survival (MTT conversion relative to thenegative/solvent control). The potential impact on melanin productionwas evaluated by determining the concentration of melanin produced inthe test article-treated tissues compared to the negative/solventcontrol-treated tissues. Data was presented in the form of concentrationof melanin produced by the test article-treated tissues determined usinga melanin standard curve. Alternatively, data may be presented aspercent change in melanin concentration relative to the negative/solventcontrol-treated tissues.

The methods used are a modification of the procedures supplied by MatTekCorporation.

Media and Reagents

MelanoDerm™ Maintenance Medium (EPI-100-LLMM) was purchased from MatTekCorporation. MelanoDerm™ Skin Model (MEL-300-A) was purchased fromMatTek Corporation. 1% Kojic acid (prepared in sterile, deionized water)was purchased from Sigma MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) waspurchased from Sigma. Dulbecco's Modified Eagle's Medium (DMEM)containing 2 mM L-glutamine (MTT Addition Medium) was purchased fromQuality Biological. Extraction Solvent (Isopropanol) was purchased fromAldrich. Sterile Ca++ and Mg++ Free Dulbecco's Phosphate Buffered Saline(CMF-DPBS) was purchased from Invitrogen. Melanin was purchased fromSigma. Sterile deionized water was purchased from Quality Biological.Solvable was purchased from Perkin Elmer.

Preparation and Delivery of Test Article

Unless otherwise specified within this protocol, twenty five microlitersof each test article were applied directly on the tissue so as to coverthe upper surface. Depending on the nature of the test article (liquids,gels, creams, foams, etc.), the use of a dosing device, mesh or otheraid to allow the uniform spreading of the test article over the surfaceof the tissue may have been necessary.

Route of Administration

The test articles were applied topically to the MelanoDerm™ tissue every48 hours (within a timeframe of 48+2 hours from previous treatment)during a 7-day trial. Twenty five microliters of each test article wereapplied to each tissue. Twenty five microliters of the positive andnegative/solvent controls, respectively, were applied to each tissue.

pH Determination

The pH of the neat liquid test article (and/or dosing solution asappropriate) was determined, if possible. The pH was determined using pHpaper (for example, with a pH range of 0-14 to estimate, and/or a pHrange of 5-10 to determine a more precise value). The typical pHincrements on the narrower range pH paper were approximately 0.3 to 0.5pH units. The maximum increment on the pH paper was 1.0 pH units.

Controls

The definitive assay included a negative control, a positive control andone solvent control (DMSO). The MelanoDerm™ tissues designated to theassay negative control were treated with 25 μL of sterile, deionizedwater. Twenty five microliters of 1% Kojic acid (prepared in sterile,deionized water and filtered at the time of preparation) was used todose the tissues designated to the assay positive control. The 1% Kojicacid was stored in a tube covered with aluminum foil until used within 2hours of preparation. The negative/solvent and positive control exposuretimes were identical to those used for the test articles. Untreatedtissues were also used as controls.

Assessment of Direct Test Article Reduction of MTT

It was necessary to assess the ability of each test article to directlyreduce MTT. A 1.0 mg/mL MTT solution was prepared in MTT AdditionMedium. Approximately 25 μL of the test article was added to 1 mL of theMTT solution and the mixture was incubated in the dark at 37±1° C. forone to three hours. A negative control, 25 μL of sterile, deionizedwater, was tested concurrently. If the MTT solution color turnedblue/purple, the test article was presumed to have reduced the MTT.Water insoluble test materials may have shown direct reduction(darkening) only at the interface between the test article and themedium.

Receipt of MelanoDerm™

Upon receipt of the MelanoDerm™ Skin Kit, the solutions were stored asindicated by the manufacturer. The MelanoDerm™ tissues were stored at2-8° C. until used.

On the day of receiving (the day before dosing), an appropriate volumeof MelanoDerm™ Maintenance Medium (EPI-100-LLMM) was removed and warmedto 37±1° C. Nine-tenths (0.9) mL of EPI-100-LLMM/well were aliquotedinto the appropriate wells of 6-well plates. Each MelanoDerm™ tissue wasinspected for air bubbles between the agarose gel and cell cultureinsert prior to opening the sealed package. Tissues with air bubblesgreater than 50% of the cell culture insert area were not used. The24-well shipping containers were removed from the plastic bag and thesurface disinfected with 70% ethanol. An appropriate number ofMelanoDerm™ tissues were transferred aseptically from the 24-wellshipping containers into the 6-well plates. The MelanoDerm™ tissues wereincubated at 37±1° C. in a humidified atmosphere of 5±1% CO₂ in air(standard culture conditions) overnight (at least 16 hours) to acclimatethe tissues. Upon opening the bag, any unused tissues remaining on theshipping agar at the time of tissue transfer were briefly gassed with anatmosphere of 5% CO_(2/95)% air, and the bag was sealed and stored at2-8° C. for subsequent use.

Definitive Assay

Tissue Exposure: At least 16 hours after initiating the cultures, fiveMelanoDerm™ tissues (considered untreated at Day 0) were photographedusing a digital camera to aid in the visual assessment of the degree ofpigmentation of the tissues at time zero of the assay. Two MelanoDerm™tissues were rinsed with CMF-DPBS, blotted dry on sterile absorbentpaper and cleared of excess liquid. The MelanoDerm™ tissues weretransferred to the appropriate MTT containing wells after rinsing andprocessed in the MTT assay. Three MelanoDerm™ tissues were rinsed withCMF-DPBS, blotted dry on sterile absorbent paper and cleared of excessliquid. The MelanoDerm™ tissues were removed from the cell cultureinsert using sterile scalpels, placed in a labeled 1.5 mL microfugetube, and stored at <−60° C. for subsequent melanin analysis.

At least 16 hours after initiating the cultures, the rest of the tissueswere transferred on a new 6-well plate containing 0.9 mL/well of fresh,pre-warmed EPI-100-LLMM. The trial was conducted over a 7-day timeframe.Five tissues were treated topically on the first day, and every 48 hours(within a timeframe of 48+2 hours from previous treatment) with 25 μL,of each test article. The medium was refreshed daily (within a timeframeof 24+2 hours from previous refeeding); the tissues were transferred toa new 6-well plate containing 0.9 mL/well of fresh, pre-warmedEPI-100-LLMM.

Five tissues were treated topically on the first day, and every 48 hours(within a timeframe of 48+2 hours from previous treatment) with 25 μL ofpositive and negative/solvent controls, respectively. The medium wasrefreshed daily (within a timeframe of 24+2 hours from previousrefeeding); the tissues were transferred to a new 6-well platecontaining 0.9 mL/well of fresh, pre-warmed EPI-100-LLMM. The tissueswere incubated at 37±1° C. in a humidified atmosphere of 5±1% CO₂ in air(standard culture conditions) for the appropriate exposure times.

On the days of dosing, the MelanoDerm™ tissue was first gently rinsedthree times using ˜500 μL of CMF-DPBS per rinse to remove any residualtest article. The CMF-DPBS was gently pipetted into the well and thendrawn off with a sterile aspirator. The tissues were transferred to anew 6-well plate containing 0.9 mL of fresh, pre-warmed EPI-100-LLMM anddosed with the appropriate test article, negative/solvent or positivecontrol. The tissues were incubated at 37±1° C. in a humidifiedatmosphere of 5±1% CO₂ in air (standard culture conditions) for theappropriate exposure times.

At the end of the 7-day trial, the MelanoDerm™ tissues treated with thenegative/solvent or positive control, and with each test article werephotographed using a digital camera to aid in the visual assessment ofthe degree of pigmentation of the tissues at the end of the assay (Day7). Then, the viability of two tissues treated with the positive andnegative control, respectively, and with each test article, weredetermined by MTT reduction. At the end of the 7-day trial, the melaninproduced by three tissues treated with each test article, the positiveand negative/solvent control, respectively, was determined.

MTT Assay: A 10× stock of MTT prepared in PBS (filtered at time of batchpreparation) was thawed and diluted in warm MTT Addition Medium toproduce the 1.0 mg/mL solution no more than two hours before use. Threehundred μL of the MTT solution was added to each designated well of aprelabelled 24-well plate.

After the exposure time, each MelanoDerm™ tissue designated for the MTTassay was rinsed with CMF-DPBS (use of spray bottle acceptable for thisstep), blotted dry on sterile absorbent paper, and cleared of excessliquid. The MelanoDerm™ tissues were transferred to the appropriate MTTcontaining wells after rinsing. The 24-well plates were incubated atstandard conditions for 3±0.1 hours.

After 3±0.1 hours, the MelanoDerm™ tissues were blotted on sterileabsorbent paper, cleared of excess liquid, and transferred to aprelabelled 24-well plate containing 2.0 mL of isopropanol in eachdesignated well. The plates were covered with parafilm and stored in therefrigerator (2-8° C.) until the last exposure time was harvested. Ifnecessary, plates were stored overnight (or up to 24 hours after thelast exposure time is harvested) in the refrigerator prior to extractingthe MTT. Then the plates were shaken for at least 2 hours at roomtemperature. At the end of the extraction period, the liquid within thecell culture inserts was decanted into the well from which the cellculture insert was taken. The extract solution was mixed and 200 μLtransferred to the appropriate wells of 96-well plate. Two hundred μL ofisopropanol was added to the wells designated as blanks. The absorbanceat 550 nm (OD550) of each well was measured with a Molecular DevicesVmax plate reader.

Melanin Assay: At the end of the appropriate exposure times, theMelanoDerm™ tissues designated for the melanin assay were gently rinsedat least three times using ˜500 μL of CMF-DPBS per rinse to remove anyresidual test article or excess phenol red from culture medium, blotteddry on sterile absorbent paper and cleared of excess liquid. TheMelanoDerm™ tissues were photographed using a digital camera at the endof the assay. The MelanoDerm™ tissues were removed from the cell cultureinsert using sterile scalpels or sterile punche(s), placed in a labeled1.5 mL microfuge tube, and stored at <−60° C. for subsequent melaninanalysis.

On the day of the melanin extraction assay, the excised tissues werethawed at room temperature for approximately 10 minutes. 250 μL Solvablewas added to each microfuge tube and the tubes were incubated for atleast 16 hours at 60+2° C. A 1 mg/mL Melanin standard stock solution wasprepared by dissolving the Melanin in Solvable. A series of Melaninstandards was prepared from the 1 mg/mL stock ranging from 0 mg/mL to0.33 mg/mL. The standard series was prepared by adding 0.6 mL of the 1mg/mL Melanin standard stock solution to 1.2 mL Solvable, and thenmaking a series of five more dilutions (dilution factor of 3). Solvablewas used as the zero standard. The Melanin standards series and theSolvable were incubated for at least 16 hours at 60+2° C.

At least 16 hours after initiating the melanin extraction, the tubescontaining the samples (representing the melanin extracted from theMelanoDerm™ tissues) and the standards were cooled at room temperatureand centrifuged at 13,000 rpm for 5 minutes at room temperature. 200 μLof samples (single wells) or standards (duplicate wells) weretransferred to the appropriate wells of a 96-well plate. Two hundred μLof Solvable were added to the wells designated as blanks in duplicatewells. The absorbance at 490 nm (OD490) of each well was measured with aMolecular Devices Vmax plate reader (with Automix function selected).

Killed Controls for Assessment of Residual Test Article Reduction of MTT

To demonstrate that possible residual test article was not acting todirectly reduce the MTT, a functional check was performed in thedefinitive assay to show that the test material was not binding to thetissue and leading to a false MTT reduction signal.

To determine whether residual test article was acting to directly reducethe MTT, a freeze-killed control tissue was used. Freeze killed tissuewas prepared by placing untreated MelanoDerm™/EpiDerm™ (Melanoderm™without melanocytes) tissues in the −20° C. freezer at least overnight,thawing to room temperature, and then refreezing. Once killed, thetissue may be stored indefinitely in the freezer. Freeze killed tissuesmay be received already prepared from MatTek Corporation, and stored inthe −20° C. freezer until use. To test for residual test articlereduction, killed tissues were treated with the test article in thenormal fashion. All assay procedures were performed in the same manneras for the viable tissue. At least one killed control treated withsterile deionized water (negative killed control) was tested in parallelsince a small amount of MTT reduction is expected from the residual NADHand associated enzymes within the killed tissue.

If little or no MTT reduction was observed in the test article-treatedkilled control, the MTT reduction observed in the test article-treatedviable tissue may be ascribed to the viable cells. If there wasappreciable MTT reduction in the treated killed control (relative to theamount in the treated viable tissue), additional steps must be taken toaccount for the chemical reduction or the test article may be considereduntestable in this system.

Data Analysis

The mean OD550 value of the blank wells was calculated. The correctedmean OD550 value of the negative/solvent control(s) was determined bysubtracting the mean OD550 value of the blank wells from their meanOD550 values. The corrected OD550 values of the individual test articleexposures and the positive control exposures was determined bysubtracting from each the mean OD550 value for the blank wells. Allcalculations were performed using an Excel spreadsheet. Although thealgorithms discussed are performed to calculate the final endpointanalysis at the treatment group level, the same calculations can beapplied to the individual replicates.

Corr. Test article exposure OD ₅₅₀=Test article exposure OD ₅₅₀−Blankmean OD ₅₅₀

If killed controls (KC) were used, the following additional calculationswere performed to correct for the amount of MTT reduced directly by testarticle residues. The raw OD550 value for the negative control killedcontrol was subtracted from the raw OD550 values for each of the testarticle-treated killed controls, to determine the net OD550 values ofthe test article-treated killed controls.

Net OD550 for each test article KC=Raw OD550 test article KC−Raw OD ₅₅₀negative/solvent control KC

The net OD550 values represent the amount of reduced MTT due to directreduction by test article residues at specific exposure times. Ingeneral, if the net OD550 value is greater than 0.150, the net amount ofMTT reduction will be subtracted from the corrected OD550 values of theviable treated tissues to obtain a final corrected OD550 value. Thesefinal corrected OD550 values will then be used to determine the % ofControl viabilities.

Final Corrected OD550=Corrected test article OD550 (viable)−Net OD550test article (KC)

Finally, the following % of Control calculations will be made:

% Viability=[(Final corrected OD ₅₅₀ of Test Article or PositiveControl)/(Corrected mean OD ₅₅₀ of Negative/Solvent Control(s))]×100

Melanin Analysis: The raw absorbance data was captured, saved as aprint-file and imported into an Excel spreadsheet. The OD490 value ofeach test sample (representing the melanin extracted from untreatedMelanoDerm™ tissues at Day 0, MelanoDerm™ tissues treated with each testarticle, negative/solvent or positive controls at Day 7) and of themelanin standards was determined. The corrected OD490 value for the testsamples and each melanin standard was determined by subtracting the meanOD490 value of the blank wells. The standard curve was plotted as theconcentration of the standards in mg/mL (y-axis) versus thecorresponding corrected absorbance. The amount of melanin in eachindividual tissue was interpolated from the standard curve (linear).Finally, the average of melanin concentration for each test article orcontrol treatment groups, respectively, was calculated.

Results

FIG. 1 summarizes the mean tissue viability and melanin concentrationresults for the test articles, positive control, and untreated tissues.Preliminary results suggest that certain formulations applied to thecarbazole compounds of the present invention may independently exhibitmoderate skin brightening effects that dampen the skin darkeningactivity of the carbazoles.

FIG. 2 summarizes the mean tissue viability and melanin concentrationresults for the test articles and untreated tissues observed in aseparate experiment. Combination treatments comprising, for example,malassezin and indirubin, exhibited more effective skin brighteningeffects than either compound on its own.

Example 6 Melanogenesis Potential of Indirubin and Indirubin Derivatives

The purpose of this study is to observe and report melanogenesis andviability of B16 melanocytes exposed to indirubin and indirubinderivatives.

Materials and Reagents

Plating media will include DMEM without L-glutamine, FBS,penicillin/streptomycin, and L-glutamine. Assay media will include DMEMwithout phenol red and L-glutamine, FBS, penicillin/streptomycin,L-glutamine, and aMSH. Other reagents will include Kojic Acid, DMSO, andMTT. Cells tested will be B16 cells (ATCC CRL-6475).

Protocol

B16 Melanocytes are cultured until 70% confluent and harvested. Cellsare seeded in 96-well plates at a density of 4000 cells/well and areallowed to attach overnight. The following day, test articles andcontrols are diluted in B16 Assay media. Overnight media is aspiratedand 200 ul of test articles and controls are applied. Cells areincubated at 37° C. and 10% CO₂ for 72 hours. Following 72-hourincubation, absorbance is read at 540 nm. Media is removed and replacedwith 100 ul of plating media containing 1 mg/mL MTT and incubated for 2hours at 37° C. and 10% CO₂. MTT media is removed and replaced with 200ul of 95% Ethanol/5% Isopropanol and allowed to shake for 15 minutes.MTT absorbance then is read at 570 nm.

Results

It is expected that the compounds and compositions of the presentinvention, including indirubin and chemical analogs thereof, willinhibit melanogenesis. Chemical analogs of indirubin are expected toexhibit, for example, more potent melanogenesis-inhibiting activitycompared to indirubin. Likewise, certain chemical analogs of indirubinare expected to demonstrate, for example, less effectivemelanogenesis-inhibiting activity compared to indirubin.

Example 7 In Vitro Efficacy

It is expected that the compounds and compositions of the presentinvention will induce melanocyte apoptosis and modulate melanocyteactivity, melanin production, melanosome biogenesis, and/or melanosometransfer at least as potently as indirubin. It is also contemplated thatcertain of the compounds and compositions of the present invention willaffect these biological processes less potently than indirubin. Suchcompounds and compositions may have more favorable toxicity profilescompared to more potent species.

Example 8 In Vivo Efficacy

It is expected that the compounds and compositions of the presentinvention will be at least as effective as indirubin for modulating skinpigmentation, including brightening skin, and improvinghyperpigmentation/hypopigmentation caused by various disorders. It isfurther expected that the compounds and compositions of the presentinvention will exhibit favorable pharmacokinetic profiles in terms of,for example, half-life and absorption. Certain compounds will exhibit alonger half-life, whereas others will exhibit a shorter half-life.Similarly, certain compounds will exhibit different absorption profiles,with some compounds taking longer to be fully absorbed and others takingless time to be fully absorbed.

Example 9 Apoptosis-Inducing Activity of Compositions ContainingMalassezia—Derived Compounds and/or Chemical Analogs Thereof Reagents

Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit, Fetal Bovine Serum(FBS), 0.25% Trypsin-EDTA (lx), Caspase-Glo 3/7 Assay, RPMI 1640 Medium,Dulbecco's Modified Eagle Medium, and Antibiotic Antimycotic Solution(100×).

The cell lines MeWo (ATCC® HTB-65™), WM115 (ATCC® CRL-1675) and B16F1(ATCC® CRL-6323) are maintained in the following culture media: culturemedium for MeWo and B16F1: DMEM supplemented with 10% FBS; culturemedium for WM115: RPMI 1640 supplemented with 10% FBS.

Experimental Methods

Cells are harvested and the cell number determined using a Countess CellCounter. The cells are diluted with culture medium to the desireddensity. The final cell density may be, for example, 4,000 cells/wellfor 6 hr and 24 hr treatment, and 2,000 cells/well for 48 hr and 72 hrtreatment. For the Annexin V assay, 384-well clear-bottom plates(Corning 3712) are employed, whereas 384-well solid white-bottom plates(Corning 3570) are used for the Caspase-Glo assays. All plates arecovered with a lid and placed at 37° C. and 5% CO₂ overnight for cellattachment.

Test compounds are dissolved in DMSO to 30 mM stock. 10-fold dilutionsare performed to generate 3 mM and 0.3 mM concentrations. 0.9 mMStaurosporine is employed as positive control, and DMSO is employed asnegative control (NC). 132.5 nL of compounds is transferred fromcompound source plate to 384-well cell culture plate(s) using liquidhandler Echo550. After the indicated incubation time, the plates areremoved from the incubator for detection.

Test compositions are dissolved DMSO, EPI-100-LLMM, or any appropriatesolvent and may be prepared according to the instructions in Tables 2-7below. Appropriate solvents are well known to those of skill in the art.

For the Annexin V assay, plates are removed from the incubator andculture media is removed. Cells are washed twice with 40 uL PBS and 15uL of pre-mixed Annexin V-FITC and Hoechst 33342 dye working solutionare added per well. Plates are incubated at room temperature for 20minutes, sealed, and centrifuged for 1 minute at 1,000 rpm to removebubbles. Plates are read using ImageXpress Nano.

For the Caspase-Glo assay, plates are removed from the incubator andequilibrated at room temperature for 15 minutes. Caspase-Glo 3/7reagents also are thawed and equilibrated to room temperature before theexperiment. Caspase-Glo reagent is added to the required wells at 1:1ratio to the culture medium. Plates are incubated at room temperaturefor 15 minutes and read using EnSpire™ plate reader. Fold induction iscalculated according to the following formula: Foldinduction=Lum_(Sample)/Lum_(NC).

Annexin V Assay and Caspase 3/7 Assay Results

It is expected that the compounds and compositions of the presentinvention, including Compositions #1-5, will induce cell death.Compositions of the present invention are expected to exhibit, forexample, more potent apoptosis-inducing activity compared to at leastone component compound alone. Likewise, compositions of the presentinvention are expected to demonstrate, for example, less effectiveapoptosis-inducing activity compared to at least one component compoundalone. Such compositions may have more favorable toxicity profilescompared to more potent compositions.

Example 10 Cell Viability After Exposure to Compositions ContainingMalassezia—Derived Compounds and/or Chemical Analogs Thereof Reagents

CellTiter-Glo® 2.0 assay.

Experimental Methods

For the CellTiter-Glo assay, test compounds are prepared in 10 mM DMSOsolution. Compounds are serially diluted into 12 concentrations. 40 uLof cells from a 100,000 cell/mL suspension are dispensed into each wellof a 384-well plate (Corning 3570). Plates are incubated overnight at37° C., 5% CO₂, and 95% humidity. Test compounds are added, with DMSO asvehicle control. Plates are incubated at 37° C., 5% CO₂, and 95%humidity for 6, 24, or 48 hours, and 40 uL of CellTiter-Glo reagent isadded to the wells to assess cell viability.

Test compositions are dissolved DMSO, EPI-100-LLMM, or any appropriatesolvent and may be prepared according to the instructions in Tables 4-9below. Appropriate solvents are well known to those of skill in the art.

Results

It is expected that the compounds and compositions of the presentinvention, including Compositions #1-5, will induce cell death.Compositions of the present invention are expected to exhibit, forexample, more potent apoptosis-inducing activity compared to at leastone component compound alone. Likewise, compositions of the presentinvention are expected to demonstrate, for example, less effectiveapoptosis-inducing activity compared to at least one component compoundalone. Such compositions may have more favorable toxicity profilescompared to more potent compositions.

Example 11 Arylhydrocarbon Receptor Activation Potential of CompositionsContaining Malassezia—Derived Compounds and/or Chemical Analogs ThereofAssay Procedures

Culture media for stably transfected HepG2 cells is prepared bysupplementing DMEM with high glucose and L-glutamine, as well as 10%FBS.

HepG2-AhR-Luc cells are cultured in T-75 flasks at 37° C., 5% CO₂, and95% relative humidity. Cells are allowed to reach 80-90% confluencebefore detachment and splitting.

Cultivated cells are rinsed with 5 mL PBS. PBS is aspirated away, 1.5 mLtrypsin is added to the flask, and cells are incubated at 37° C. forapproximately 5 minutes or until the cells are detached and float.Trypsin is inactivated by adding excess serum-containing media.

The cell suspension is transferred to a conical tube and centrifuged at120 g for 10 minutes to pellet the cells. Cells are resuspended inseeding media at a proper density. 40 μL of cells are transferred to a384-well culture plate (5×10³ cells/well). Plates are placed in theincubator at 37° C. for 24 hours.

Afterward, stock solutions of test compounds, test compositions, andomeprazole positive control are prepared. Compound and compositionssolutions are transferred into the assay plate using Echo550. The plateis then placed back into the incubator for compound/compositiontreatment.

Later, after 24 hours of treatment, the plate is removed from theincubator and allowed to cool at ambient temperature. 30 μL One-Gloreagent equal to that of the culture medium is added in each well. Cellsare allowed to lyse for at least 3 minutes, and then measured in aluminometer.

Dose responses are graphed using the non-linear regression analysis inXLfit, and EC₅₀ values are also calculated.

Results

It is expected that the compounds and compositions of the presentinvention, including Compositions #1-5, will modulate AhR activity.Compositions of the present invention are expected to exhibit, forexample, more potent AhR agonist activity compared to at least onecomponent compound alone. Likewise, compositions of the presentinvention are expected to demonstrate, for example, less effective AhRagonist activity compared to at least one component compound alone.Compositions of the present invention also are expected to exhibit, forexample, more potent AhR antagonist activity compared to at least onecomponent compound alone. Likewise, compositions of the presentinvention also are expected to demonstrate, for example, less effectiveAhR antagonist activity compared to at least one component compoundalone.

Example 12 MelanoDerm™ Assays

The purpose of this study was to evaluate the potential action of thetest articles as a skin melanogenesis modulator in the MelanoDerm™ SkinModel after repeated test article exposures. Secondarily, the purpose ofthis study was to evaluate the potential dermal irritation of the testarticle to the MelanoDerm™ Skin Model after repeated exposures. Toxicitywas determined by measuring the relative conversion of MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in thetest article-treated tissues compared to the negative/solventcontrol-treated tissues. The potential impact on melanin production wasdetermined by measuring the concentration of melanin produced by thetest article-treated tissues compared to the negative/solventcontrol-treated tissues.

Identification of Test Substances and Assay Controls

TABLE 4 Test Articles Tested in Diluted Form Test Article Sponsor DosingDesignation Designation Concentration Preparation Instructions 18AH47DMSO 0.5% (v/v) The solvent control was diluted (v/v) with EPI- (solvent100-LLMM to a final concentration of 0.5%; the control) diluted solventcontrol was vortexed for at least 1 minute and dosed onto the tissuesusing a dosing volume of 25 μL. A total volume of up to 0.5 mL wasprepared for each tissue treatment. 17AJ41 Malassezin 500 μM Startingfrom the stock concentration provided by (CV-8684) the Sponsor/preparedfrom the solid material (Positive provided by the Sponsor, the testarticle/control control) was diluted (v/v) with EPI-100-LLMM to the17AJ55 O52 650 μM dosing concentration listed. The test article 18AA21Malassezia 650 μM dilution was vortexed for at least 1 minute, heatedIndole A at 37° ± 1° C. (in a water bath) for 15 minutes, 18AF50 AB17151300 μM vortexed again for at least 1 minute and dosed on 18AH15 AB17590300 μM the tissues using a dosing volume of 25 μL. A 18AH21 AB11644 650μM total volume of up ~0.5 mL was prepared for 18AH38 Indole-3- 500 μMeach tissue treatment. carbaldehyde 18AH39 D-indole-3- 500 μM lacticacid

TABLE 5 Composition #1 Preparation Preparation Instructions ForInstructions For Dilutions Used Test Article Sponsor Working StockDosing For Dosing of Designation Designation Solutions Concentration theTissues 17AD42 Indolo-carbazole A working stock The dosing Fifty (50) μLof (ICZ) solution of 360 μM concentration of each working 17AJ41Malassezin was prepared each of the stock solution was (CV-8684) fromthe top stock components was transferred into a (Positive control)solution in DMSO 18 μM. new vial 17AJ47 Compound A5 as follows: The(combined (also known as stock solution was volume of 700 μL)Keto-Malassezin) thawed at room and mixed 17AJ55 O52 temperature andwith 300 μL of 18AA21 Malassezia Indole A vortexed for ~1 EPI-100-LLMM18AA22 Pityriacitrin minute. The to yield a total 18AA24 FICZappropriate volume of 1000 μL. 18AD42 Indirubin volume needed to Thedilution 18AH16 Trypthantrin prepare up to ~0.5 mL/ was vortexed for18AH20 Malassezia-lactic 1.0 mL of at least 1 minute Acid working stockbefore being 18AH24 2-hydroxy-1-(1H- solution was applied onto theindol-3- transferred to a tissues. yl)ethanone new vial and 18AH38Indole-3- diluted with EPI- carbaldehyde 100-LLMM to 18AH39D-Indole-3-lactic 360 μM. The acid dilution was 18AH44 (Indol-3-vortexed for at yl)pyruvic acid least 1 minute, heated at 37° ± 1° C.(in a water bath) for 15 minutes and vortexed again for at least 1minute before being subsequently diluted.

TABLE 6 Composition #2 Preparation Preparation Instructions ForInstructions For Volume Dilutions Used Test Article Sponsor WorkingStock Dosing Needed For Dosing of Designation Designation SolutionsConcentration (μL) the Tissues 17AD42 Indolo- A working stock 12.6 μM 35The volume of carbazole (ICZ) solution of 360 μM the dosing 17AJ41Malassezin was prepared 50.4 μM 140 concentration (CV-8684) from the topstock listed for each (Positive solution in DMSO component was control)as follows: The transferred into 17AJ47 Compound A5 stock solution was10.1 μM 28 a new vial and (also known as thawed at room mixed with 297μL Keto- temperature and of EPI-100- Malassezin) vortexed for ~1 LLMM.The 17AJ55 O52 minute. The 10.1 μM 28 dilution was 18AA21 Malasseziaappropriate 10.1 μM 28 vortexed for at Indole A volume needed to least 1minute 18AA22 Pityriacitrin prepare up to ~0.5 mL/ 50.4 μM 140 beforebeing 18AA24 FICZ 1.0 mL of 10.1 μM 28 applied onto the 18AD42 Indirubinworking stock 24.5 μM 68 tissues. 18AH16 Trypthantrin solution was 24.5μM 68 18AH20 Malassezia- transferred to a 10.1 μM 28 lactic Acid newvial and 18AH24 2-hydroxy-1- diluted with EPI- 10.1 μM 28 (1H-indol-3-100-LLMM to 360 μM. yl)ethanone The dilution 18AH38 Indole-3- wasvortexed for at 10.1 μM 28 carbaldehyde least 1 minute, 18AH39D-Indole-3- heated at 37° ± 1° C. 10.1 μM 28 lactic acid (in a waterbath) 18AH44 (Indol-3- for 15 minutes and 10.1 μM 28 yl)pyruvic acidvortexed again for at least 1 minute before being subsequently diluted.

TABLE 7 Composition #3 Preparation Preparation Instructions forInstructions for Dosing Volume Dilutions Used Test Article SponsorWorking Stock Concentration Needed for Dosing of Designation DesignationSolutions (μM) (μL) the Tissues 17AJ41 Malassezin A working stock 50.4140 The volume of (CV-8684) solution of 360 μM the dosing (Positive wasprepared from concentration control) the top stock listed for each17AD46 Compound A5 solution in DMSO 10.1 28 component (CV-8819) asfollows: The was transferred (also known as stock solution was into anew vial Keto- thawed at room and mixed Malassezin) temperature and with568 μL of 17AJ55 O52 vortexed for ~1 10.1 28 EPI-100- (AB12976) minute.The LLMM. The 18AA21 Malassezia appropriate 10.1 28 dilution was IndoleA volume needed to vortexed for at (AB17011) prepare up to ~0.5 mL/least 1 minute 18AD42 Indirubin 1.0 mL of 24.5 68 before being 18AH20AB17227 working stock 10.1 28 applied onto (also known as solution wasthe tissues. Malassezia- transferred to a lactic Acid) new vial and18AH24 2-hydroxy-1- diluted with EPI- 10.1 28 (1H-indol-3- 100-LLMM to360 μM. yl)ethanone The dilution 18AH38 Indole-3- was vortexed for at10.1 28 carbaldehyde least 1 minute, 18AH39 D-Indole-3- heated at 37° ±1° C. 10.1 28 lactic acid (in a water bath) 18AH44 (Indol-3- for 15minutes and 10.1 28 yl)pyruvic acid vortexed again for at least 1 minutebefore being subsequently diluted.

TABLE 8 Composition #4 Preparation Preparation Instructions forInstructions for Dosing Volume Dilutions Used Test Article SponsorWorking Stock Concentration Needed for Dosing of Designation DesignationSolutions (μM) (μL) the Tissues 17AD42 CV-8685 A working 12.6 35 Thevolume of (also known as stock solution the dosing Indolo- of 360 μM wasconcentration carbazole or prepared from listed for each ICZ) the topstock component was 17AJ41 Malassezin solution in DMSO 50.4 140transferred (CV-8684) as follows: The into a new vial (Positive stocksolution and mixed control) was thawed at with 505 μL of 17AD46 CompoundA5 room temperature 10.1 28 EPI-100- (CV-8819) and vortexed LLMM. The(also known as for ~1 minute. dilution was Keto- The appropriatevortexed for at Malassezin) volume needed least 1 minute 17AJ55 O52 toprepare up 10.1 28 before being (AB12976) to ~0.5 mL/1.0 applied onto18AA21 Malassezia mL of working 10.1 28 the tissues. Indole A stocksolution (AB17011) was transferred to 18AA24 FICZ a new vial and 10.1 2818AD42 Indirubin diluted with 24.5 68 18AH20 AB17227 EPI-100-LLMM 10.128 (also known as to 360 μM. The Malassezia- dilution was lactic Acid)vortexed for at 18AH24 2-hydroxy-1- least 1 minute, 10.1 28 (1H-indol-3-heated at yl)ethanone 37° ± 1° C. (in a 18AH38 Indole-3- water bath) for10.1 28 carbaldehyde 15 minutes and 18AH39 D-Indole-3- vortexed again10.1 28 lactic acid for at least 1 18AH44 (Indol-3- minute before 10.128 yl)pyruvic acid being subsequently diluted.

TABLE 9 Composition #5 Preparation Preparation Instructions forInstructions for Dosing Volume Dilutions Used Test Article SponsorWorking Stock Concentration Needed for Dosing of Designation DesignationSolutions (μM) (μL) the Tissues 17AD42 CV-8685 A working 74.9 208 Thevolume of (also known as stock solution the dosing Indolo- of 360 μM wasconcentration carbazole or prepared from listed for each ICZ) the topstock component was 17AJ41 Malassezin solution in 10.1 28 transferred(CV-8684) DMSO as into a new vial (Positive follows: The and mixedcontrol) stock solution with 306 μL of 18AA22 Pityriacitrin was thawedat 10.1 28 EPI-100- (AB17014) room temperature LLMM. The 18AA24 FICZ andvortexed 74.9 208 dilution was 18AD42 Indirubin for ~1 minute. 24.8 69vortexed for at 18AH16 Trypthantrin The appropriate 10.1 28 least 1minute 18AH24 2-hydroxy-1- volume needed 10.1 28 before being(1H-indol-3- to prepare up applied onto yl)ethanone to ~0.5 mL/1.0 thetissues. 18AH39 D-Indole-3- mL of working 24.8 69 lactic acid stocksolution 18AH44 (Indol-3- was transferred 10.1 28 yl)pyruvic acid to anew vial and diluted with EPI-100-LLMM to 360 μM. The dilution wasvortexed for at least 1 minute, heated at 37° ± 1° C. (in a water bath)for 15 minutes and vortexed again for at least 1 minute before beingsubsequently diluted.

Assay controls include: positive control—malassezin (CV-8684) (500 μM)(17AJ41) and solvent control—DMSO (dimethyl sulfoxide) prepared inEPI-100-LLMM.

Additionally, the test article and controls were applied to groups of 4tissues of which 2 were used for the Tissue Viability (MTT) endpoint and2 for the Melanin endpoint, respectively.

Test System

The MelanoDerm™ Skin Model provided by MatTek Corporation (Ashland,Mass.) was used in this study. The MelanoDerm™ tissue consists ofnormal, human-derived epidermal keratinocytes (NHEK) and melanocytes(NHM) which have been cultured to form a multilayered, highlydifferentiated model of the human epidermis. The NHMs within co-culturesundergo spontaneous melanogenesis leading to tissues of varying levelsof pigmentation. The cultures were grown on cell culture inserts at theair-liquid interface, allowing for topical application of skinmodulators. The MelanoDerm™ model exhibits in vivo-like morphologicaland ultrastructural characteristics. NHM localized in the basal celllayer of MelanoDerm™ tissue are dendritic and spontaneously producemelanin granules which progressively populate the layers of the tissue.Thus the test system is used to screen for materials which may inhibitor stimulate the production of melanin relative to the negativecontrols.

Experimental Design and Methodology

The experimental design of this study consisted of the determination ofthe pH of the neat test article if possible (and/or dosing solution asappropriate) and a definitive assay to determine the relative tissueviability and the potential action of the test article as a skinmelanogenesis modulator to MelanoDerm™ Skin Model after repeatedexposures. The test articles were exposed to the MelanoDerm™ Skin Modelfor a total of 7 days. The test articles were topically applied to theMelanoDerm™ Skin Model every 48 hours (within a timeframe of 48±2 hoursfrom previous treatment). The toxicity of the test articles weredetermined by the NAD(P)H-dependent microsomal enzyme reduction of MTT(and, to a lesser extent, by the succinate dehydrogenase reduction ofMTT) in control and test article-treated tissues. Data was presented inthe form of relative survival (MTT conversion relative to thenegative/solvent control). The potential impact on melanin productionwas evaluated by determining the concentration of melanin produced inthe test article-treated tissues compared to the negative/solventcontrol-treated tissues. Data was presented in the form of concentrationof melanin produced by the test article-treated tissues determined usinga melanin standard curve. Alternatively, data may be presented aspercent change in melanin concentration relative to the negative/solventcontrol-treated tissues.

The methods used are a modification of the procedures supplied by MatTekCorporation.

Media and Reagents

MelanoDerm™ Maintenance Medium (EPI-100-LLMM) was purchased from MatTekCorporation. MelanoDerm™ Skin Model (MEL-300-A) was purchased fromMatTek Corporation. 1% Kojic acid (prepared in sterile, deionized water)was purchased from Sigma MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) waspurchased from Sigma. Dulbecco's Modified Eagle's Medium (DMEM)containing 2 mM L-glutamine (MTT Addition Medium) was purchased fromQuality Biological. Extraction Solvent (Isopropanol) was purchased fromAldrich. Sterile Ca++ and Mg++ Free Dulbecco's Phosphate Buffered Saline(CMF-DPBS) was purchased from Invitrogen. Melanin was purchased fromSigma. Sterile deionized water was purchased from Quality Biological.Solvable was purchased from Perkin Elmer.

Preparation and Delivery of Test Article

Unless otherwise specified within this protocol, twenty five microlitersof each test article were applied directly on the tissue so as to coverthe upper surface. Depending on the nature of the test article (liquids,gels, creams, foams, and the like), the use of a dosing device, mesh orother aid to allow the uniform spreading of the test article over thesurface of the tissue may have been necessary.

Route of Administration

The test articles were applied topically to the MelanoDerm™ tissue every48 hours (within a timeframe of 48+2 hours from previous treatment)during a 7-day trial. Twenty five microliters of each test article wereapplied to each tissue. Twenty five microliters of the positive andnegative/solvent controls, respectively, were applied to each tissue.

pH Determination

The pH of the neat liquid test article (and/or dosing solution asappropriate) was determined, if possible. The pH was determined using pHpaper (for example, with a pH range of 0-14 to estimate, and/or a pHrange of 5-10 to determine a more precise value). The typical pHincrements on the narrower range pH paper were approximately 0.3 to 0.5pH units. The maximum increment on the pH paper was 1.0 pH units.

Controls

The definitive assay included a negative control, a positive control andone solvent control (DMSO) or a positive control and a solvent control(DMSO). The MelanoDerm™ tissues designated to the assay negative/solventcontrol were treated with 25 μL of sterile, deionized water or DMSO. Thetissues designated to the assay positive control were treated with 25 μLof 1% Kojic acid, Malassezin (CV-8684) (17AJ41) 500 μM, or Composition#2. The 1% Kojic acid was stored in a tube covered with aluminum foiluntil used within 2 hours of preparation. The negative/solvent andpositive control exposure times were identical to those used for thetest articles. Untreated tissues were also used as controls.

Assessment of Direct Test Article Reduction of MTT

It was necessary to assess the ability of each test article to directlyreduce MTT. A 1.0 mg/mL MTT solution was prepared in MTT AdditionMedium. Approximately 25 μL of the test article was added to 1 mL of theMTT solution and the mixture was incubated in the dark at 37±1° C. forone to three hours. A negative control, 25 μL of sterile, deionizedwater, or a solvent control, 25 μL of DMSO was tested concurrently. Ifthe MTT solution color turned blue/purple, the test article was presumedto have reduced the MTT. Water insoluble test materials may have showndirect reduction (darkening) only at the interface between the testarticle and the medium.

Receipt of MelanoDerm™

Upon receipt of the MelanoDerm™ Skin Kit, the solutions were stored asindicated by the manufacturer. The MelanoDerm™ tissues were stored at2-8° C. until used.

On the day of receiving (the day before dosing), an appropriate volumeof MelanoDerm™ Maintenance Medium (EPI-100-LLMM) was removed and warmedto 37±1° C. Nine-tenths (0.9) mL of EPI-100-LLMM/well were aliquotedinto the appropriate wells of 6-well plates. Each MelanoDerm™ tissue wasinspected for air bubbles between the agarose gel and cell cultureinsert prior to opening the sealed package. Tissues with air bubblesgreater than 50% of the cell culture insert area were not used. The24-well shipping containers were removed from the plastic bag and thesurface disinfected with 70% ethanol. An appropriate number ofMelanoDerm™ tissues were transferred aseptically from the 24-wellshipping containers into the 6-well plates. The MelanoDerm™ tissues wereincubated at 37±1° C. in a humidified atmosphere of 5±1% CO₂ in air(standard culture conditions) overnight (at least 16 hours) to acclimatethe tissues. Upon opening the bag, any unused tissues remaining on theshipping agar at the time of tissue transfer were briefly gassed with anatmosphere of 5% CO_(2/95)% air, and the bag was sealed and stored at2-8° C. for subsequent use.

Definitive Assay

Tissue Exposure: At least 16 hours after initiating the cultures, fiveMelanoDerm™ tissues (considered untreated at Day 0) were photographedusing a digital camera to aid in the visual assessment of the degree ofpigmentation of the tissues at time zero of the assay. Two MelanoDerm™tissues were rinsed with CMF-DPBS, blotted dry on sterile absorbentpaper and cleared of excess liquid. The MelanoDerm™ tissues weretransferred to the appropriate MTT containing wells after rinsing andprocessed in the MTT assay. Two or three MelanoDerm™ tissues were rinsedwith CMF-DPBS, blotted dry on sterile absorbent paper and cleared ofexcess liquid. The MelanoDerm™ tissues were removed from the cellculture insert using sterile scalpels, placed in a labeled 1.5 mLmicrofuge tube, and stored at <−60° C. for subsequent melanin analysis.

At least 16 hours after initiating the cultures, the rest of the tissueswere transferred on a new 6-well plate containing 0.9 mL/well of fresh,pre-warmed EPI-100-LLMM. The trial was conducted over a 7-day timeframe.Four or five tissues were treated topically on the first day, and every48 hours (within a timeframe of 48+2 hours from previous treatment) with25 μL, of each test article. The medium was refreshed daily (within atimeframe of 24+2 hours from previous refeeding); the tissues weretransferred to a new 6-well plate containing 0.9 mL/well of fresh,pre-warmed EPI-100-LLMM.

Four or five tissues were treated topically on the first day, and every48 hours (within a timeframe of 48+2 hours from previous treatment) with25 μL of positive and negative/solvent controls, respectively. Themedium was refreshed daily (within a timeframe of 24+2 hours fromprevious refeeding); the tissues were transferred to a new 6-well platecontaining 0.9 mL/well of fresh, pre-warmed EPI-100-LLMM. The tissueswere incubated at 37±1° C. in a humidified atmosphere of 5±1% CO₂ in air(standard culture conditions) for the appropriate exposure times.

On the days of dosing, the MelanoDerm™ tissue was first gently rinsedthree times using ˜500 μL of CMF-DPBS per rinse to remove any residualtest article. The CMF-DPBS was gently pipetted into the well and thendrawn off with a sterile aspirator. The tissues were transferred to anew 6-well plate containing 0.9 mL of fresh, pre-warmed EPI-100-LLMM anddosed with the appropriate test article, negative/solvent or positivecontrol. The tissues were incubated at 37±1° C. in a humidifiedatmosphere of 5±1% CO₂ in air (standard culture conditions) for theappropriate exposure times.

At the end of the 7-day trial, the MelanoDerm™ tissues treated with thenegative/solvent or positive control, and with each test article werephotographed using a digital camera to aid in the visual assessment ofthe degree of pigmentation of the tissues at the end of the assay (Day7). Then, the viability of two tissues treated with the positive andnegative control, respectively, and with each test article, weredetermined by MTT reduction. At the end of the 7-day trial, the melaninproduced by three tissues treated with each test article, the positiveand negative/solvent control, respectively, was determined.

MTT Assay: A 10× stock of MTT prepared in PBS (filtered at time of batchpreparation) was thawed and diluted in warm MTT Addition Medium toproduce the 1.0 mg/mL solution no more than two hours before use. Threehundred μL of the MTT solution was added to each designated well of aprelabelled 24-well plate.

After the exposure time, each MelanoDerm™ tissue designated for the MTTassay was rinsed with CMF-DPBS (use of spray bottle acceptable for thisstep), blotted dry on sterile absorbent paper, and cleared of excessliquid. The MelanoDerm™ tissues were transferred to the appropriate MTTcontaining wells after rinsing. The 24-well plates were incubated atstandard conditions for 3±0.1 hours.

After 3±0.1 hours, the MelanoDerm™ tissues were blotted on sterileabsorbent paper, cleared of excess liquid, and transferred to aprelabelled 24-well plate containing 2.0 mL of isopropanol in eachdesignated well. The plates were covered with parafilm and stored in therefrigerator (2-8° C.) until the last exposure time was harvested. Ifnecessary, plates were stored overnight (or up to 24 hours after thelast exposure time is harvested) in the refrigerator prior to extractingthe MTT. Then the plates were shaken for at least 2 hours at roomtemperature. At the end of the extraction period, the liquid within thecell culture inserts was decanted into the well from which the cellculture insert was taken. The extract solution was mixed and 200 μLtransferred to the appropriate wells of 96-well plate. Two hundred μL ofisopropanol was added to the wells designated as blanks. The absorbanceat 550 nm (OD550) of each well was measured with a Molecular DevicesVmax plate reader.

Melanin Assay: At the end of the appropriate exposure times, theMelanoDerm™ tissues designated for the melanin assay were gently rinsedat least three times using ˜500 μL of CMF-DPBS per rinse to remove anyresidual test article or excess phenol red from culture medium, blotteddry on sterile absorbent paper and cleared of excess liquid. TheMelanoDerm™ tissues were photographed using a digital camera at the endof the assay. The MelanoDerm™ tissues were removed from the cell cultureinsert using sterile scalpels or sterile punche(s), placed in a labeled1.5 mL microfuge tube, and stored at <−60° C. for subsequent melaninanalysis.

On the day of the melanin extraction assay, the excised tissues werethawed at room temperature for approximately 10 minutes. 250 μL Solvablewas added to each microfuge tube and the tubes were incubated for atleast 16 hours at 60+2° C. A 1 mg/mL Melanin standard stock solution wasprepared by dissolving the Melanin in Solvable. A series of Melaninstandards was prepared from the 1 mg/mL stock ranging from 0 mg/mL to0.33 mg/mL. The standard series was prepared by adding 0.6 mL of the 1mg/mL Melanin standard stock solution to 1.2 mL Solvable, and thenmaking a series of five more dilutions (dilution factor of 3). Solvablewas used as the zero standard. The Melanin standards series and theSolvable were incubated for at least 16 hours at 60+2° C.

At least 16 hours after initiating the melanin extraction, the tubescontaining the samples (representing the melanin extracted from theMelanoDerm™ tissues) and the standards were cooled at room temperatureand centrifuged at 13,000 rpm for 5 minutes at room temperature. 200 μLof samples (single wells) or standards (duplicate wells) weretransferred to the appropriate wells of a 96-well plate. Two hundred μLof Solvable were added to the wells designated as blanks in duplicatewells. The absorbance at 490 nm (OD490) of each well was measured with aMolecular Devices Vmax plate reader (with Automix function selected).

Killed Controls for Assessment of Residual Test Article Reduction of MTT

To demonstrate that possible residual test article was not acting todirectly reduce the MTT, a functional check was performed in thedefinitive assay to show that the test material was not binding to thetissue and leading to a false MTT reduction signal.

To determine whether residual test article was acting to directly reducethe MTT, a freeze-killed control tissue was used. Freeze killed tissuewas prepared by placing untreated MelanoDerm™/EpiDerm™ (Melanoderm™without melanocytes) tissues in the −20° C. freezer at least overnight,thawing to room temperature, and then refreezing. Once killed, thetissue may be stored indefinitely in the freezer. Freeze killed tissuesmay be received already prepared from MatTek Corporation, and stored inthe −20° C. freezer until use. To test for residual test articlereduction, killed tissues were treated with the test article in thenormal fashion. All assay procedures were performed in the same manneras for the viable tissue. At least one killed control treated withsterile deionized water (negative killed control) was tested in parallelsince a small amount of MTT reduction is expected from the residual NADHand associated enzymes within the killed tissue.

If little or no MTT reduction was observed in the test article-treatedkilled control, the MTT reduction observed in the test article-treatedviable tissue may be ascribed to the viable cells. If there wasappreciable MTT reduction in the treated killed control (relative to theamount in the treated viable tissue), additional steps must be taken toaccount for the chemical reduction or the test article may be considereduntestable in this system.

Data Analysis

The mean OD550 value of the blank wells was calculated. The correctedmean OD550 value of the negative/solvent control(s) was determined bysubtracting the mean OD550 value of the blank wells from their meanOD550 values. The corrected OD550 values of the individual test articleexposures and the positive control exposures was determined bysubtracting from each the mean OD550 value for the blank wells. Allcalculations were performed using an Excel spreadsheet. Although thealgorithms discussed are performed to calculate the final endpointanalysis at the treatment group level, the same calculations can beapplied to the individual replicates.

Corr. Test article exposure OD550=Test article exposure OD550−Blank meanOD550

If killed controls (KC) were used, the following additional calculationswere performed to correct for the amount of MTT reduced directly by testarticle residues. The raw OD550 value for the negative control killedcontrol was subtracted from the raw OD550 values for each of the testarticle-treated killed controls, to determine the net OD550 values ofthe test article-treated killed controls.

Net OD ₅₅₀ for each test article KC=Raw OD ₅₅₀ test article KC−Raw OD₅₅₀ negative/solvent control KC

The net OD550 values represent the amount of reduced MTT due to directreduction by test article residues at specific exposure times. Ingeneral, if the net OD550 value is greater than 0.150, the net amount ofMTT reduction will be subtracted from the corrected OD550 values of theviable treated tissues to obtain a final corrected OD550 value. Thesefinal corrected OD550 values will then be used to determine the % ofControl viabilities.

Final Corrected OD550=Corrected test article OD550 (viable)−Net OD550test article (KC)

Finally, the following % of Control calculations will be made:

% Viability=[(Final corrected OD ₅₅₀ of Test Article or PositiveControl)/(Corrected mean OD ₅₅₀ of Negative/Solvent Control(s))]×100

Melanin Analysis: The raw absorbance data was captured, saved as aprint-file and imported into an Excel spreadsheet. The OD490 value ofeach test sample (representing the melanin extracted from untreatedMelanoDerm™ tissues at Day 0, MelanoDerm™ tissues treated with each testarticle, negative/solvent or positive controls at Day 7) and of themelanin standards was determined. The corrected OD490 value for the testsamples and each melanin standard was determined by subtracting the meanOD490 value of the blank wells. The standard curve was plotted as theconcentration of the standards in mg/mL (y-axis) versus thecorresponding corrected absorbance. The amount of melanin in eachindividual tissue was interpolated from the standard curve (linear).Finally, the average of melanin concentration for each test article orcontrol treatment groups, respectively, was calculated.

Results

FIG. 4 summarizes the mean tissue viability and melanin concentrationresults for the test articles, test compositions, positive control, andsolvent control. The compounds comprising compositions #1 and #2demonstrated synergistic effects when combined in a single composition.

FIG. 5 summarizes the mean tissue viability and melanin concentrationresults for the test articles, test compositions, positive control, andsolvent control. The compounds comprising compositions #2, #3, #4, and#5 demonstrated synergistic effects when combined in a singlecomposition.

Example 13 Melanogenesis Potential of Compositions ContainingMalassezia—Derived Compounds and/or Chemical Analogs Thereof

The purpose of this study is to observe and report melanogenesis andviability of B16 melanocytes exposed to compositions containingMalassezia-derived compounds and/or chemical analogs thereof.

Materials and Reagents

Plating media will include DMEM without L-glutamine, FBS,penicillin/streptomycin, and L-glutamine. Assay media will include DMEMwithout phenol red and L-glutamine, FBS, penicillin/streptomycin,L-glutamine, and aMSH. Other reagents will include Kojic Acid, DMSO, andMTT. Cells tested will be B16 cells (ATCC CRL-6475).

Protocol

B16 Melanocytes are cultured until 70% confluent and harvested. Cellsare seeded in 96-well plates at a density of 4000 cells/well and areallowed to attach overnight. The following day, test articles, testcompositions, and controls are diluted in B16 Assay media. Overnightmedia is aspirated and 200 ul of test articles and controls are applied.Cells are incubated at 37° C. and 10% CO₂ for 72 hours. Following72-hour incubation, absorbance is read at 540 nm. Media is removed andreplaced with 100 ul of plating media containing 1 mg/mL MTT andincubated for 2 hours at 37° C. and 10% CO₂. MTT media is removed andreplaced with 200 ul of 95% Ethanol/5% Isopropanol and allowed to shakefor 15 minutes. MTT absorbance then is read at 570 nm.

Results

It is expected that the compounds and compositions of the presentinvention, including Compositions #1-5, will inhibit melanogenesis.Compositions of the present invention are expected to exhibit, forexample, more potent melanogenesis-inhibiting activity compared to atleast one component compound. Likewise, certain compositions areexpected to demonstrate, for example, less effectivemelanogenesis-inhibiting activity compared to at least one componentcompound.

Example 14 In Vitro Efficacy

It is expected that the compounds and compositions of the presentinvention will induce melanocyte apoptosis and modulate melanocyteactivity, melanin production, melanin concentration, melanosomebiogenesis, and/or melanosome transfer. It is also contemplated thatcertain of the compounds and compositions of the present invention willaffect these biological processes less potently. Such compounds andcompositions may have more favorable toxicity profiles compared to morepotent species.

Example 15 In Vivo Efficacy

It is expected that the compounds and compositions of the presentinvention will modulate skin pigmentation, including brightening skin,and improving hyperpigmentation/hypopigmentation caused by variousdisorders. It is further expected that the compounds and compositions ofthe present invention will exhibit favorable pharmacokinetic profiles interms of, for example, half-life and absorption. Certain compounds willexhibit a longer half-life, whereas others will exhibit a shorterhalf-life. Similarly, certain compounds will exhibit differentabsorption profiles, with some compounds taking longer to be fullyabsorbed and others taking less time to be fully absorbed.

Example 16 Synthesis of Chemical Analogs of Malassezin and IndirubinSynthesis of AB17590

As shown in FIG. 6A, to a solution of compound 1a (25.0 g, 0.357 mol,1.0 eq) in tetrahydrofuran (250 mL) was added ethynylmagnesium bromide(0.5 M in THF, 1.07 L, 0.535 mol, 1.5 eq) at 0° C. and the reactionmixture was warmed to room temperature and stirred for 2 h. Then themixture was quenched with saturated aqueous of ammonium chloride andextracted with ethyl acetate. The organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The residue was purifiedby silica gel chromatography (0-10% ethyl acetate in petroleum ether) togive compound 1b (9.5 g, 27%). TLC: PE:EA=20:1, 254 nm; R_(f) (Compound1a)=0.3; R_(f) (Compound 1b)=0.7.

To a mixture of compound 1b (9.5 g, 98.96 mmol, 1.0 eq) intetrahydrofuran (100 mL) was added a solution of 60% sodium hydride (4.7g, 0.119 mol, 1.2 eq) in dimethylformamide (50 mL) at 0° C. undernitrogen atmosphere. After 30 minutes, dimethyl sulphate (22.4 g, 0.178mol, 1.8 eq) was added at 0° C. After the addition the reaction mixturewas allowed to warm to room temperature and stirred at room temperaturefor 30 min and then acetic acid (1 ml) was added slowly. The product wasdistilled directly from the reaction mixture. There was thus obtainedcompound 1c (10.0 g, 91% yield).

To a solution of compound 1 (8.0 g, 24.02 mmol, 1.0 eq) and compound 1c(2.9 g, 26.43 mmol, 1.1 eq) in triethylamine (80 mL) was added cuprousiodide (456 mg, 2.40 mmol, 0.1 eq) and Pd(PPh₃)₂Cl₂ (337 mg, 0.480 mmol,0.02 eq) at room temperature under nitrogen atmosphere. The mixture wasstirred at room temperature for 2 h. The progress of the reactionmixture was monitored by TLC. The reaction mixture was diluted withwater and extracted with ethyl acetate. The organic layer was dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography (0˜10% ethyl acetatein petroleum ether) to give compound 2 (7.0 g, 92%). TLC: PE: EA=10:1,254 nm; R_(f) (compound 1)=0.8; R_(f) (compound 2)=0.6.

To an oven-dried flask was added a mixture of platinum dichloride (694mg, 2.06 mmol, 0.1 eq), sodium carbonate (3.3 g, 30.95 mmol, 1.5 eq),tris (pentafluorophenyl) phosphine (2.2 g, 4.13 mmol, 0.2 eq), 6-methylindole (4.8 g, 41.27 mmol, 2.0 eq) and compound 2 (6.5 g, 20.63 mmol,1.0 eq) in dioxane (650 mL). The flask was degassed with nitrogen,sealed and heated to 100° C. for 16 h. The progress of the reactionmixture was monitored by TLC. The solvent was concentrated under reducedpressure. The residue was diluted with ethyl acetate and extracted withwater, saturated brine. The organic layer was dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by silica gel chromatography (0-10% ethyl acetate in petroleumether) to give compound 3 (3.0 g, 36%). TLC: PE: EA=10:1, 254 nm; R_(f)(compound 2)=0.6; R_(f) (compound 3)=0.2.

To a solution of compound 3 (3.0 g, 7.50 mmol, 1.0 eq) intetrahydrofuran (30 mL) was added sodium methanolate (5 M in MeOH, 6.0mL, 29.98 mmol, 4.0 eq) at 0° C. The reaction mixture was allowed towarm to room temperature and stirred for 2 h. The progress of thereaction mixture was monitored by TLC. The reaction mixture wasconcentrated under reduced pressure. The residue was purified by silicagel chromatography (0-10% ethyl acetate in petroleum ether) to givecompound 4 (1.5 g, 66%). TLC: PE: EA=5:1, 254 nm; R_(f) (compound3)=0.7; R_(f) (compound 4)=0.4.

To a dried 500 mL three-neck round-bottom flask under argon at 0° C.,dimethylformamide (10 mL) was added. Then phosphorus oxychloride (1.2 g,7.60 mmol, 1.2 eq) was slowly added while maintaining the internaltemperature below 5° C. over 10 min. After stirring at 0° C. for 30 min,a solution of compound 4 (1.9 g, 6.33 mmol, 1.0 eq) in dimethylformamide(20 mL) was slowly added while maintaining the internal temperaturebelow 5° C. over 10 min. The resulting mixture was stirred at roomtemperature for 16 h. After the reaction was complete (monitored by TLCusing 20% ethyl acetate in hexanes), the reaction mixture was pouredinto saturated aqueous sodium bicarbonate (50 mL) and stirred for 1 h.Resulting mixture was extracted with ethyl acetate (2×100 mL). Thecombined organic layers were washed with water, saturated brine anddried over sodium sulfate. The solvent was filtered and concentratedunder reduced pressure. The residue was purified by silica gelchromatography (10-50% ethyl acetate in petroleum ether) to obtaincompound 5 (1.8 g, 89%). TLC: PE: EA=1:1, 254 nm; R_(f) (compound4)=0.8; R_(f) (compound 5)=0.5.

To a solution of compound 5 (1.8 g, 5.49 mmol, 1.0 eq) intetrahydrofuran (20 mL) was added Di-tert-butyl dicarbonate (3.0 g,13.72 mmol, 2.5 eq) and 4-Dimethylaminopyridine (1.4 g, 11.25 mol, 2.05eq) at 0° C. The reaction mixture was warmed to room temperature andstirred for 3 h. The progress of the reaction mixture was monitored byTLC. The reaction mixture was concentrated under reduced pressure andthe residue was diluted with ethyl acetate and washed with 1Nhydrochloric acid, saturated aqueous sodium bicarbonate (300 mL) andbrine (300 mL). The organic layers were separated and dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel chromatography (0-10% ethyl acetate in petroleumether) to obtain compound 6 (2.4 g, 82%). TLC: PE: EA=10:1, 254 nm;R_(f) (compound 5)=0.1; R_(f) (compound 6)=0.5.

To a solution of compound 6 (2.4 g, 4.55 mmol, 1.0 eq) in tert-Butanol(60 mL) was added 2-methyl-2-butene (30 mL) followed by addition ofsodium chlorite (8.2 g, 90.91 mmol, 20.0 eq), sodium phosphate monobasic(14.2 g, 90.91 mmol, 20.0 eq) and water (60 mL) at 0° C. The mixture wasslowly warmed to room temperature and stirred at room temperature for 15h. The progress of the reaction mixture was monitored by TLC. Thereaction mixture was diluted with dichloromethane (100 mL) andseparated. The organic layer was washed with water (80 mL), brine (80mL), dried over anhydrous sodium sulfate and concentrated under reducedpressure to obtain crude compound 7 (2.5 g, 99%). TLC: PE: EA=2:1, 254nm; R_(f) (compound 6)=0.7; R_(f) (compound 7)=0.3.

To a solution of compound 7 (2.5 g, 4.60 mmol, 1.0 eq) indimethylformamide (30 mL) was added potassium carbonate (952 mg, 6.89mmol, 1.5 eq) and methyl iodide (978 mg, 6.89 mmol, 1.5 eq) at 0° C. Thereaction mixture was warmed to room temperature and stirred for 2 h. Theprogress of the reaction mixture was monitored by TLC. The reactionmixture was diluted with ethyl acetate (100 mL) and washed with water(100 mL) and brine (100 mL). The organic layer was dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by silica gel chromatography (5-17% ethyl acetate in petroleumether) to obtain compound 8 (2.3 g, 89%). TLC: PE: EA=5:1, 254 nm; R_(f)(compound 7)=0.1; R_(f) (compound 8)=0.6.

A mixture of compound 8 (1.3 g, 2.33 mmol, 1.0 eq) in hydrochloric acid(3 M in EA, 30 mL) was stirred at room temperature for 16 h. Thereaction was monitored by TLC. Then the mixture was concentrated underreduced pressure. The residue was purified by silica gel chromatography(10-25% ethyl acetate in petroleum ether) to give compound AB17590 (502mg, 61%) as a yellow solid. TLC: PE: EA=3:1, 254 nm; R_(f) (compound8)=0.8; R_(f) (compound AB17590)=0.5; LC-MS: 359 (M+1)+; ¹H NMR (400MHz, CDCl₃) δ 8.12 (d, J=19.7 Hz, 2H), 7.94 (s, 1H), 7.42 (s, 1H), 7.35(d, J=8.1 Hz, 1H), 7.13 (t, J=7.8 Hz, 1H), 7.04 (d, J=8.2 Hz, 1H), 6.93(dd, J=15.7, 8.6 Hz, 2H), 5.04 (d, J=9.1 Hz, 1H), 3.95 (s, 3H), 2.45 (s,3H), 1.42 (d, J=8.4 Hz, 1H), 0.78-0.68 (m, 1H), 0.62 (d, J=4.8 Hz, 1H),0.54-0.41 (m, 2H).

Synthesis of AB17653

As shown in FIG. 6B, a mixture of compound 1 (721 mg, 3.20 mmol, 1.0eq), compound 1a (560 mg, 3.20 mmol, 1.0 eq) and sodium carbonate (866mg, 8.17 mmol, 2.55 eq) in methanol (10 mL) was stirred at roomtemperature for 3 h under nitrogen atmosphere. The progress of thereaction mixture was monitored by TLC. After completion of the reaction,the mixture was filtered and the filter cake was washed with methanoland water to afford compound AB17653 (979 mg, 89%) as a red solid. TLC:PE/EA=3/1, 254 nm; R_(f) (Compound 1)=0.6; R_(f) (Compound AB17653)=0.4;LC-MS: 338.95 (M−1)⁻; ¹H NMR (400 MHz, d6-DMSO) δ11.01 (d, J=21.5 Hz,2H), 8.64 (d, J=8.3 Hz, 1H), 7.62 (d, J=7.7 Hz, 1H), 7.55 (t, J=7.6 Hz,1H), 7.39 (d, J=8.1 Hz, 1H), 7.18 (d, J=8.4 Hz, 1H), 7.00 (dd, J=8.8,4.6 Hz, 2H).

Synthesis of AB17654

As shown in FIG. 6B, a mixture of compound AB17653 (979 mg, 2.88 mmol,1.0 eq) and hydroxylamine hydrochloride (520 mg, 7.49 mmol, 2.6 eq) inpyridine (30 mL) was stirred at 120° C. for 2 h under nitrogenatmosphere. The progress of the reaction mixture was monitored by LCMS.After completion of the reaction, the mixture was concentrated underreduced pressure and added 1 N HCl until the solid appeared. The mixturewas filtered and the filter cake was dissolved in 1 N NaOH. Then 3 N HClwas added to adjust pH=5 and filtered. The filter cake was washed with 1N HCl to afford compound AB17654 (500 mg, 48%) as a red solid. LC-MS:357.95 (M+1)⁺; ¹H NMR (400 MHz, d6-DMSO) δ 13.59 (s, 1H), 11.71 (s, 1H),10.82 (s, 1H), 8.53 (d, J=8.4 Hz, 1H), 8.19 (d, J=7.7 Hz, 1H), 7.42-7.35(m, 2H), 7.11-6.96 (m, 3H).

Synthesis of AB17655

As shown in FIG. 6B, a mixture of compound 2 (637 mg, 3.86 mmol, 1.0eq), compound 1a (676 mg, 3.86 mmol, 1.0 eq) and sodium carbonate (1044mg, 9.84 mmol, 2.55 eq) in methanol (10 mL) was stirred at roomtemperature for 3 h under nitrogen atmosphere. The progress of thereaction mixture was monitored by TLC. After completion of the reaction,the mixture was filtered and the filter cake was washed with methanoland water to afford compound AB17655 (1027 mg, 95%) as a red solid.LC-MS: 281.05 (M+1)⁺; ¹H NMR (400 MHz, d6-DMSO) δ11.06 (s, 1H), 10.86(s, 1H), 8.54 (dd, J=10.5, 2.7 Hz, 1H), 7.67-7.53 (m, 2H), 7.41-7.38 (m,1H), 7.09-6.98 (m, 2H), 6.85 (dd, J=8.5, 4.8 Hz, 1H).

Synthesis of AB17656

As shown in FIG. 6B, a mixture of compound AB17655 (1027 mg, 3.67 mmol,1.0 eq) and hydroxylamine hydrochloride (663 mg, 9.54 mmol, 2.6 eq) inpyridine (30 mL) was stirred at 110° C. for 2 h under nitrogenatmosphere. The progress of the reaction mixture was monitored by LCMS.After completion of the reaction, the mixture was concentrated underreduced pressure and added 1 N HCl until the solid appeared. The mixturewas filtered and the filter cake was dissolved in 1 N NaOH. Then 3 N HClwas added to adjust pH=5 and filtered. The filter cake was washed with 1N HCl to afford compound AB17656 (500 mg, 48%) as a red solid. LC-MS:296.00 (M+1)⁺; ¹H NMR (400 MHz, d6-DMSO) δ13.60 (s, 1H), 11.77 (s, 1H),10.69 (s, 1H), 8.43 (s, 1H), 8.20 (d, J=7.7 Hz, 1H), 7.39 (d, J=5.7 Hz,2H), 7.02 (s, 1H), 6.91 (s, 1H), 6.83 (d, J=4.9 Hz, 1H).

Synthesis of AB17657

As shown in FIG. 6B, a mixture of compound 3 (362 mg, 2.46 mmol, 1.0eq), compound 1a (431 mg, 2.46 mmol, 1.0 eq) and sodium carbonate (666mg, 6.28 mmol, 2.55 eq) in methanol (10 mL) was stirred at roomtemperature for 3 h under nitrogen atmosphere. The progress of thereaction mixture was monitored by TLC. After completion of the reaction,the mixture was filtered and the filter cake was washed with methanoland water to afford compound 4 (606 mg, 93%). TLC: PE/EA=1/1, 254 nm;R_(f) (Compound 3)=0.7; R_(f) (Compound 4)=0.5.

A mixture of compound 4 (606 mg, 2.31 mmol, 1.0 eq) and hydroxylaminehydrochloride (418 mg, 6.01 mmol, 2.6 eq) in pyridine (20 mL) wasstirred at 120° C. for 2 h under nitrogen atmosphere. The progress ofthe reaction mixture was monitored by TLC. After completion of thereaction, the mixture was concentrated under reduced pressure and added1 N HCl until the solid appeared. The mixture was filtered and thefilter cake was dissolved in 1 N NaOH. Then 3 N HCl was added to adjustpH=5 and filtered. The filter cake was washed with 1 N HCl to affordcompound AB17657 (500 mg, 78%) as a brown solid. TLC: PE/EA=1/1, 254 nm;R_(f) (Compound 4)=0.5; R_(f) (Compound AB17657)=0.4; LC-MS: 278.10(M+1)⁺; ¹H NMR (400 MHz, d6-DMSO) δ13.60 (s, 1H), 11.77 (s, 1H), 10.69(s, 1H), 8.43 (s, 1H), 8.20 (d, J=7.7 Hz, 1H), 7.39 (d, J=5.7 Hz, 2H),7.02 (s, 1H), 6.91 (s, 1H), 6.83 (d, J=4.9 Hz, 1H).

Synthesis of AB17658

As shown in FIG. 6B, a mixture of compound 5a (337 mg, 1.73 mmol, 1.0eq), compound 5b (554 mg, 1.73 mmol, 1.0 eq) and potassium hydroxide(1114 mg, 3.46 mmol, 2.0 eq) in acetonitrile (10 mL) was stirred at 35°C. for 1.5 h under nitrogen atmosphere. The progress of the reactionmixture was monitored by TLC. After completion of the reaction, themixture was concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford compound 5c (436 mg,99%). TLC: PE/EA=1/1, 254 nm; R_(f) (Compound 5a)=0.8; R_(f) (Compound5c)=0.5.

A mixture of compound 5 (330 mg, 1.72 mmol, 1.0 eq), compound 5c (436mg, 1.72 mmol, 1.0 eq) and sodium carbonate (465 mg, 4.38 mmol, 2.55 eq)in methanol (10 mL) was stirred at room temperature for 3 h undernitrogen atmosphere. The progress of the reaction mixture was monitoredby TLC. After completion of the reaction, the mixture was filtered andthe filter cake was washed with methanol and water to afford compound 6(617 mg, 93%). TLC: PE/EA=1/1, 254 nm; R_(f) (Compound 5)=0.5; R_(f)(Compound 6)=0.4.

A mixture of compound 6 (617 mg, 1.60 mmol, 1.0 eq) and hydroxylaminehydrochloride (290 mg, 4.17 mmol, 2.6 eq) in pyridine (20 mL) wasstirred at 110° C. for 2 h under nitrogen atmosphere. The progress ofthe reaction mixture was monitored by TLC. After completion of thereaction, the mixture was concentrated under reduced pressure and added1 N HCl until the solid appeared. The mixture was filtered and thefilter cake was dissolved in 1 N NaOH. Then 3 N HCl was added to adjustpH=5 and filtered. The filter cake was washed with 1 N HCl to affordcompound AB17658 (500 mg, 78%) as a red solid. TLC: PE/EA=1/1, 254 nm;R_(f) (Compound 6)=0.4; R_(f) (Compound AB17658)=0.3; LC-MS: 402.95(M+1)⁺; ¹H NMR (400 MHz, d6-DMSO) δ11.86 (s, 1H), 11.39 (s, 1H), 9.40(d, J=2.2 Hz, 1H), 8.33 (d, J=1.8 Hz, 1H), 8.06 (dd, J=8.6, 2.4 Hz, 1H),7.59 (dd, J=8.4, 2.0 Hz, 1H), 7.43 (d, J=8.6 Hz, 1H), 7.02 (d, J=8.6 Hz,1H).

Example 17 In Vivo Assessment of the Photoprotective Properties ofMalassezin, Other Malassezia-Derived Compounds, and Chemical AnalogsThereof Malassezin 1% Formulation

The Malassezin 1% formulation used in this study contained the followingingredients: Water (aqua)—65.939%; Dimethyl isosorbide—20.000%; OliveOil Glycereth-8 Esters—3.000%; Glycerin—2.991%; Coconut Alkanes—2.700%;Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer—1.700%;Malassezin—1.000%; Pentylene Glycol—1.000%; Phenoxyethanol—0.640%;Coco-Caprylate/Caprate—0.300%; Caprylyl Glycol—0.200%;Chlorphenesin—0.160%; Sorbitan Isostearate—0.140%; Tocopherol—0.100%;Polysorbate 60-0.080%; and Disodium EDTA—0.050%.

Experimental Design

A 39-year-old Skin Type IV female was included in this Proof of Conceptstudy.

On Day 1 of the experiment, the subject was evaluated to determineMinimal Erythema Dosing (“IVIED”) using a targeted broad band DualightUVB device. A template of 6 squares was placed on the lower left back(1.5 cm×1.5 cm) of the test subject. See FIG. 7.

The MED photo test doses for the subject's skin type are listed in FIG.8 in mJ/cm² units. Twenty-four hours after irradiation, the subjectreturned for MED assessment. As shown in FIG. 12, the subject's MED was120 mJ.

Subsequently, the subject applied Malassezin 1% in the superior testsquare of the right back twice daily for 7 days. A second right lowersquare was treated twice daily from day 4 to day 7, and a third medialsquare for one application on day 7. The product vehicle was applied for7 days twice daily on the left back. See FIG. 13. The subject returnedto the research center for irradiation on day 7. See FIG. 9. Each testsite was irradiated with 120 mJ of UVB exposure. The subject returned in24 hours for assessment of phototoxicity/photoprotection. See FIG. 14.

The subject continued the experiment, receiving Malassezin 1% for atotal of 14 days. FIGS. 15-16 show regions of the subject's skin exposedto the following treatments: on site 14, Malassezin 1% was applied twicea day for 14 days; on site 10, Malassezin 1% was applied twice a day for11 days; on site 8, Malassezin 1% was applied twice a day for 8 days; onsite 3, Malassezin 1% was applied twice a day for 3 days; on site 1,Malassezin 1% was applied once; and, on the vehicle sites, vehicle wasapplied twice a day for 7 and 9 days, respectively.

Results

As shown in FIG. 14, 24 hours after UVB exposure, the subject exhibited1 plus to 2 plus erythema at the vehicle test site. See FIG. 11 forerythema scale. In contrast, there was less erythema (mild) noted at theMalassezin 1% 7-day treatment site. Evaluation of sites treated for 3days showed minimal erythema and none for the 1-day application site.Colorimetry measurements were taken from each site using the MexameterMX16 and supported clinical observations. Maximal erythema readings wereobserved in the vehicle site followed by the Malassezin 7-day-treatedsite. The lowest values were observed for the Malassezin day 3 and day 1site, respectively. See FIG. 9.

The subject continued the experiment and returned for a repeat UVBirradiation at 14 days with interpretation at day 15. See FIG. 15.Clinical evaluation at day 15 revealed moderate erythema at the vehiclesite for day 7 and significantly less at day 9. See FIG. 16. Lesserythema (mild) was noted at the Malassezin 1%-treated sites, includingthe day 14, day 10, and day 8 sites Minimal erythema was noted atMalassezin 1% sites for days 1 and day 3. Colorimetry readings weretaken from each site to measure erythema and the melanin index. Resultssupported clinical observations of less erythema at the Malassezin1%-treated sites. See FIG. 10.

Biopsies were taken from the vehicle site at 9 days and the Malassezin1%-treated sites for days 1 and 3. Specimens were analyzed forHematoxylin and Eosin, Fontana Masson staining and MART I forquantification of melanocytes and affymetrix studies.

Diagnosis: (A) Skin—Day 1 Treated (Malassezin 1%): Basket weave stratumcorneum, normal appearing melanocytes (confirmed by immunoperoxidasestaining with Mart-1), and epidermal melanin (confirmed byimmunoperoxidase staining with Fontana Masson).

Diagnosis: (B) Skin—Day 3 Treated (Malassezin 1%): Basket weave stratumcorneum, less dendritic melanocytes (confirmed by immunoperoxidasestaining with MART-1/Melan A) when compared to C and D, and with aslight decrease in epidermal melanin, as skip areas (confirmed byimmunoperoxidase staining with Fontana Masson).

Diagnosis: (C) Skin—Vehicle: Normal appearing epidermal melanocytes(confirmed by immunoperoxidase staining with Mart-1) and epidermalmelanin (confirmed by immunoperoxidase staining with Fontana Masson).

Diagnosis: (D) Skin—Normal: Normal appearing epidermal melanocytes(confirmed by immunoperoxidase staining with Mart-1) and epidermalmelanin (confirmed by immunoperoxidase staining with Fontana Masson).

CONCLUSIONS

The results of this Proof of Concept study demonstrate the UV-protectiveproperties of Malassezin.

It is envisioned that further studies involving additional patients willdemonstrate equivalent or more effective UV-protective properties ofMalassezin. It also is envisioned that additional studies will elucidatemolecular signaling pathways associated with Malassezin-inducedphotoprotection.

DOCUMENTS

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All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

What is claimed is:
 1. A composition comprising one or more compoundsselected from the group consisting of:

or a crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof, and a cosmetically or pharmaceuticallyacceptable vehicle, diluent, or carrier.
 2. The composition of claim 1,wherein the composition comprises a compound selected from the groupconsisting of:

or a crystalline form, hydrate, or pharmaceutically or cosmeticallyacceptable salt thereof.
 3. The composition of claim 1, wherein thecomposition comprises a compound selected from the group consisting of:

or a pharmaceutically or cosmetically acceptable salt thereof.
 4. Thecomposition of claim 1, wherein the composition comprises a compoundselected from the group consisting of:

or a pharmaceutically or cosmetically acceptable salt thereof.
 5. Thecomposition of claim 1, wherein the composition comprises a compound ofthe following structure:

or a pharmaceutically or cosmetically acceptable salt thereof.
 6. Thecomposition of claim 32, wherein the composition comprises a compound ofthe following structure:

or a pharmaceutically or cosmetically acceptable salt thereof.
 7. Thecomposition of claim 1, wherein the composition comprises a compound ofthe following structure:

or a pharmaceutically or cosmetically acceptable salt thereof.
 8. Thecomposition of claim 1, wherein the composition comprises a compound ofthe following structure:

or a pharmaceutically or cosmetically acceptable salt thereof.
 9. Thecomposition of claim 1, wherein the composition comprises a compound ofthe following structure:

or a pharmaceutically or cosmetically acceptable salt thereof.
 10. Thecomposition of claim 1, wherein the composition comprises a compound ofthe following structure:

or a pharmaceutically or cosmetically acceptable salt thereof.